Methods and compositions for bioremediation

ABSTRACT

The present invention provides isolated nucleic acid molecules that encode one or more of the enzymes required to produce PDTC. The present invention also provides isolated proteins encoded by nucleic acid molecules of the invention. In another aspect, the present invention provides methods for reducing the amount of a metal in a substrate, such as soil. In yet another aspect, the present invention provides methods for reducing the amount of carbon tetrachloride in a substrate, such as soil.

FIELD OF THE INVENTION

[0001] This invention relates to nucleic acid molecules that encode enzymes required for the biosynthesis of pyridine-2,6-bis(thiocarboxylate), and to environmental remediation methods for removing metals and carbon tetrachloride from a contaminated substrate, such as soil or water.

BACKGROUND OF THE INVENTION

[0002] Pollution of the environment is a major problem. Examples of environmental pollutants are metals (including radioactive metal isotopes) and organic molecules, such as carbon tetrachloride, that are produced as by-products from numerous industrial processes. One approach to removing pollutants from the environment is bioremediation which utilizes one or more biological organisms to physically remove and/or degrade environmental pollutants. Ex situ bioremediation techniques involve the removal of the contaminated substance, such as contaminated soil, to a treatment facility where the contaminant(s) is removed. In situ bioremediation does not require the physical removal of the contaminated substance, which is treated at the site of contamination, and so is typically cheaper than ex situ bioremediation.

[0003] The present inventors have identified and isolated a portion of the Pseudomonas stutzeri genome that encodes enzymes necessary to synthesize pyridine-2,6-bis(thiocarboxylate) (abbreviated as PDTC). PDTC chelates numerous metal ions, and the complex formed between PDTC and Cu(II) ions is capable of degrading carbon tetrachloride. This carbon tetrachloride degradation ability is unique and particularly valuable since it mediates the conversion of carbon tetrachloride to non-toxic carbon dioxide. Other known bacterial carbon tetrachloride degradation processes convert carbon tetrachloride to toxic intermediates like chloroform.

[0004] Thus, as set forth more fully herein, the present invention provides compositions and methods that are useful to degrade carbon tetrachloride in substances, such as soil or water, that are contaminated with carbon tetrachloride. The present invention also provides methods and compositions that are useful to remove metal ions from (or immobilize metal ions within) substances, such as soil or water, that are contaminated with metal ions. The methods and compositions of the invention can be utilized in bioremediation.

SUMMARY OF THE INVENTION

[0005] In accordance with the foregoing, the present invention provides isolated nucleic acid molecules that encode one, or more, or all, of the enzymes (or functional fragments thereof) required to produce PDTC. The present invention also provides isolated proteins encoded by nucleic acid molecules of the invention. Representative examples of isolated nucleic acid molecules and isolated proteins of the invention include the nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 (encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO:2), the nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:3 (encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO:4), the nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:5 (encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO:6), and the nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:7 (encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO:8). Thus, in one aspect, the present invention provides isolated nucleic acid molecules that are at least 70% identical to a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

[0006] In another aspect, the present invention provides isolated nucleic acid molecules that comprise a PDTC gene cluster as defined herein. The isolated nucleic acid molecules of the invention that comprise a PDTC gene cluster may optionally further comprise: a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:7; a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:9 (the nucleic acid sequence set forth in SEQ ID NO:9 encodes the protein consisting of the amino acid sequence set forth in SEQ ID NO:10); and a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:11 (the nucleic acid sequence set forth in SEQ ID NO:11 encodes the protein consisting of the amino acid sequence set forth in SEQ ID NO:12).

[0007] In another aspect, the present invention provides isolated nucleic acid molecules that are at least 70% identical (such as at least 80% identical, or at least 90% identical) to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:13 which is a portion of the Pseudomonas stutzeri genome that encodes all of the enzymes necessary to synthesize pyridine-2,6-bis(thiocarboxylate). The present invention also provides vectors that include one or more nucleic acid molecules of the invention, and host cells (including bacterial and plant cells) that include one or more vectors of the invention.

[0008] In another aspect, the present invention provides isolated proteins, such as isolated proteins that are at least 70% identical to one or more of the proteins consisting of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

[0009] In another aspect, the present invention provides methods for reducing the amount of a metal in a substrate, such as soil or water. In yet another aspect, the present invention provides methods for reducing the amount of carbon tetrachloride in a substrate, such as soil or water.

[0010] In yet another aspect, the present invention provides methods for immobilizing metal ions within a substrate (such as soil), the methods including the steps of: (a) contacting PDTC with a substrate to form a metal complex with the metal ion species; and (b) allowing PDTC to form a metal complex with the metal ion species thereby immobilizing the metal ion species. The methods of this aspect of the invention are useful, for example, for immobilizing metal ions in contaminated soil. The PDTC forms a complex with the metal ions to form a water-insoluble complex, or a complex which diffuses through the soil more slowly than uncomplexed metal ions.

[0011] The nucleic acid molecules of the invention can be used, for example, to genetically modify an organism (such as a microorganism or plant), to confer on the modified organism the ability to synthesize PDTC (or augment the existing ability of the organism to synthesize PDTC). The compositions and methods of the present invention are therefor useful, for example, in bioremediation, such as in situ bioremediation where one or more biological organisms are genetically modified to gain or augment the ability to remove metal ions from the environment, and/or to degrade carbon tetrachloride. In one representative example, the compositions and methods of the invention are useful to leach metals from metal ore. Some nucleic acid molecules of the invention (such as the nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:13) are useful, for example, as vectors for the transfer of valuable genetic traits between different strains of bacteria. Further, the nucleic acid molecules of the invention can be used, for example, as probes to identify related nucleic acid molecules, or to inhibit the expression (such as through antisense inhibition) of related nucleic acid molecules. The proteins of the invention are useful, for example, to enhance or otherwise modify the biosynthetic pathway in microorganisms that results in the production or inhibition of PDTC.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0013]FIG. 1 shows a map of the insert of cosmid pT31 (SEQ ID NO:13) and positions of deletions and insertions used to assess the complementation of a PDTC phenotype. Open reading frames are indicated by arrows with letter designations below; the direction of the arrows indicate the direction of transcription/translation of the putative gene. Transposon insertions are represented by vertical lines with flags. The orientation of flags indicates the orientation of the lacZ or phoA gene of the transposon. Open flags indicate insertions with neutral effect on PDTC production; filled flags indicate inserts that have an effect on PDTC production (see Table 2). The column labeled ctt lists the CCl₄ transformation activity in units of μg CCl₄ ml⁻¹ day⁻¹ for each CTN1 transconjugant containing the respective cosmid. N.D.=not detected;

[0014]FIG. 2A illustrates the chemical structure of PDTC;

[0015] FIGS. 2B-2E illustrate the chemical structures of representative PDTC metal complexes;

[0016]FIG. 3 illustrates CCl₄ transformation by PDTC at different copper concentrations. Replicate reactions were started by addition of CCl₄, and individual reactions were sacrificed at the indicated time points. A. no added reductant. Symbols: □, no added copper (2 separate experiments); ▾, 0.19 μM CuCl₂; ♦, 3 μM CuCl₂; ▪, 5 μM CuCl₂; , 13 μM CuCl₂. B. 0.5 mM sulfide (added as H₂S). Symbols: □, no added copper; ♦, 7.5 nM CuCl₂; ▪, 75 nM CuCl₂; , 13 μM CuCl₂; +, no PDTC;

[0017]FIG. 4 illustrates reaction pathway explaining products of reaction between Cu[II]:PDTC and Cl₄. Compounds shown in gray are those that have not been identified;

[0018]FIG. 5 illustrates products of reaction between Cu:PDTC and CCl₄ detected by negative ion electrospray mass spectrometry. Reactions were conducted in DMF:H₂O using 2 mM Cu:PDTC and excess CCl₄. A. Whole reaction mixture after a 2-hour incubation. B. No CCl₄ added;

[0019]FIG. 6 illustrates the EPR spectrum of PBN-trapped radicals produced in reaction of Cu:PDTC and excess ¹³CCl₄. Reactions were conducted in phosphate-buffered aqueous solution with 5 mM PDTC, 50 μM CuCl₂ and 50 μM Na₄EDTA (included to aid solubility of the Cu complex), and 100 mM PBN. A. complete reaction mixture; B. mixture lacking Cu; C. mixture lacking PDTC;

[0020]FIG. 7 illustrates the positive ion electrospray MS/MS of products of reaction between 2 mM Cu:PDTC and excess CCl₄ in the presence of 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO). A. ES+/MS spectrum of the reaction mixture in DMF:water (1:1, vol/vol). All ions except those at m/z140 and m/z142 were present in control incubations without CCl₄. DMF: N,N-dimethylformamide; TBA+: tetrabutylammonium cation. B. ES+ MS/MS daughter ion fragments from ion at m/z 142 from reaction in A. C. Daughter ion fragments from ion at m/z 142 from authentic 2,2,6,6-tetramethylpiperidine. For secondary ionization, argon gas was used;

[0021]FIG. 8 illustrates the potentiometric titration curve for pdtc titrated with a 1 N NaOH solution: [pdtc]=0.05 mM; T=25° C. and I=0.1M (NaClO₄);

[0022]FIG. 9 illustrates the stepwise protonation of pdtc;

[0023]FIG. 10 illustrates spectral changes during titration of pdtc by NaOH. I (ionic strength adjustment)=0.1 N NaClO₄: T=25.0° C.; l (path length)=1.0 cm; [pdtc]=0.275; and mM [KOH]=1.0 M: pH=(1) 1.00; (2) 1.21; (3) 1.51; (4) 2.08; (5) 2.98; (6) 4.11; (7) 4.75; (8) 5.00; (9) 5.35; (10) 5.63; (11) 5.96; (12) 6.82; (13) 12.5;

[0024]FIG. 11 illustrates the spectral changes during titration of Fe(pdtc)₂ by NaOH. I (ionic strength adjustment)=0.1 N NaClO₄: T=25.0° C.; l=1 cm; [PDTC]=0.275 mM; and [KOH]=1.0 M: (1) Free PDTC; pH=(2) 1.00 to 9.00; (3) 10.06; (4) 11.12; (5) 11.39; (6) 11.48; (7) 11.62; (8) 11.72; (9) 12.05;

[0025]FIG. 12 illustrates the graphical determination of inflection point of Fe(pdtc)₂ titrated by NaOH. I (ionic strength adjustment)=0.1 N NaClO₄; T=25.0° C.; l=1 cm; [pdtc]=0.275; and mM [KOH]=1.0 M, λ=350; and

[0026]FIG. 13 illustrates the spectra of various metal complexes and free pdtc. I=0.1 N NaClO₄: T=25.0° C.; l=1 cm; [M]=0.275 mM; and 2% HNO₃=1.0 M: (1) [FeIII(pdtc)₂]⁻¹⁻; (2) CoIII[(pdtc)₂]¹⁻; (3) NiIII[(pdtc)₂]¹⁻; (4) MnIII[(pdtc)₂]¹⁻; (5) CrIII[(pdtc)₂]¹⁻; (6) [pdtc]²⁻; (7) CuII[pdtc]¹⁻; (8) ZnII[pdtc]¹⁻.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0027] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Press, Plainsview, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art.

[0028] As used in connection with the nucleic acid molecules and proteins of the invention, the term “isolated” means a nucleic acid molecule or protein that is substantially free from cellular components that are associated with the nucleic acid molecule or protein as it is found in nature. As used in this context, the term “substantially free from cellular components” means that the nucleic acid molecule or protein is purified to a purity level of greater than 80% (such as greater than 90%, greater than 95%, or greater than 99%). Moreover, the terms “isolated nucleic acid molecule” and “isolated protein” include nucleic acid molecules and proteins, respectively, which do not naturally occur, and have been produced by synthetic means. An isolated nucleic acid molecule or isolated protein generally resolves as a single, predominant, band by gel electrophoresis, and yields a nucleic acid sequence or amino acid sequence profile consistent with the presence of a predominant nucleic acid molecule or protein.

[0029] The term “percent identity” or “percent identical” when used in connection with the nucleic acid molecules and proteins of the present invention, is defined as the percentage of nucleic acid residues in a candidate nucleic acid sequence, or the percentage of amino acid residues in a candidate protein sequence, that are identical with a subject nucleic acid sequence (such as any one of the nucleic acid sequences set forth in SEQ ID NOS:1, 3 and 5) or protein sequence, after aligning the candidate and subject sequences to achieve the maximum percent identity, and not considering any nucleic acid residue substitutions as part of the nucleic acid sequence identity. When making the comparison, the candidate nucleic acid sequence or protein sequence (which may be a portion of a larger nucleic acid sequence or protein sequence) is the same length as the subject nucleic acid sequence or protein sequence, and no gaps are introduced into the candidate nucleic acid sequence or protein sequence in order to achieve the best alignment.

[0030] For example, if a 100 base pair subject nucleic acid sequence is aligned with a 100 base pair candidate portion of a larger DNA molecule (such as a genomic clone), and 80% of the nucleic acid residues in the 100 base pair candidate portion align with the identical nucleic acid residues in the 100 base pair subject nucleic acid sequence, then the 100 base pair candidate portion of the larger DNA molecule is 80% identical to the subject nucleic acid sequence.

[0031] Nucleic acid sequence identity can be determined in the following manner. The subject nucleic acid sequence is used to search a nucleic acid sequence database, such as the GenBank database (accessible at web site http://www.ncbi.nln.nih.gov/blast/), using the program BLASTM version 2.1 (based on Altschul et al., Nucleic Acids Research 25:3389-3402 (1997)). The program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity. The default parameters of BLASTM are utilized.

[0032] Amino acid sequence identity can be determined in the following manner. The subject protein sequence is used to search a protein sequence database, such as the GenBank database (accessible at web site http://www.ncbi.nln.nih.gov/blast/), using the BLASTP program. The program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity. The default parameters of BLASTP are utilized. Default filtering is used to remove sequence homologs due to region of low complexity.

[0033] The term “vector” refers to a nucleic acid molecule, usually double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a cDNA molecule or a fragment of genomic DNA. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. A vector may contain the necessary elements that permit transcribing and translating the insert nucleic acid molecule into a polypeptide. The insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated. Many molecules of the polypeptide (if any) encoded by the insert nucleic acid molecule can thus be rapidly synthesized.

[0034] The term “PDTC gene cluster” refers to a group of nucleic acid sequences that encode enzymes necessary to synthesize PDTC and that, when introduced into a host cell (such as a plant or bacterial cell), confer on the host cell the ability to synthesize PDTC. The PDTC gene cluster includes: (1) a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:1; (2) a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:3; (3) a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:5; (4) a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:14 (the nucleic acid sequence set forth in SEQ ID NO:14 encodes the protein set forth in SEQ ID NO:15); (5) a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:16 (the nucleic acid sequence set forth in SEQ ID NO:16 encodes the protein set forth in SEQ ID NO:17); and (6) a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:18 (the nucleic acid sequence set forth in SEQ ID NO:18 encodes the protein set forth in SEQ ID NO:19).

[0035] The term “regulatory element” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences.

[0036] The term “rhizosphere” refers to the environment adjacent to the roots of a plant, regardless of the identity of the substrate (such as soil or water) in which the plant is growing.

[0037] The term “functional fragment” when used in reference to an isolated protein of the invention refers to a fragment that is a portion of the full-length protein, provided that the fragment has a biological activity that is characteristic of the corresponding full-length protein.

[0038] The term “complement” when used in connection with a nucleic acid molecule refers to the complementary nucleic acid sequence as determined by Watson-Crick base pairing. For example, the complement of the nucleic acid sequence 5′CCATG3′ is 5′CATGG3′.

[0039] In one aspect, the present invention provides isolated nucleic acid molecules that encode one, or more, or all, of the enzymes (or a functional fragment thereof) required to produce PDTC. Thus, in one aspect, the present invention provides an isolated portion of the Pseudomonas stutzeri genome including the following open reading frames (ORFs): ORF-K (SEQ ID NO:1, encoding the protein set forth in SEQ ID NO:2); ORF-N (SEQ ID NO:3, encoding the protein set forth in SEQ ID NO:4); ORF-P (SEQ ID NO:5, encoding the protein set forth in SEQ ID NO:6); ORF-C (SEQ ID NO:7, encoding the protein set forth in SEQ ID NO:8); ORF-G (SEQ ID NO:9, encoding the protein set forth in SEQ ID NO:10); ORF-H (SEQ ID NO:11, encoding the protein set forth in SEQ ID NO:12); ORF-F (SEQ ID NO:14, encoding the protein set forth in SEQ ID NO:15); ORF-J (SEQ ID NO:16, encoding the protein set forth in SEQ ID NO:17); ORF-I (SEQ ID NO:18, encoding the protein set forth in SEQ ID NO:19); ORF-A (SEQ ID NO:20, encoding the protein set forth in SEQ ID NO:21); ORF-B (SEQ ID NO:22, encoding the protein set forth in SEQ ID NO:23); ORF-D (SEQ ID NO:24, encoding the protein set forth in SEQ ID NO:25); ORF-E (SEQ ID NO:26, encoding the protein set forth in SEQ ID NO:27); ORF-L (SEQ ID NO:28, encoding the protein set forth in SEQ ID NO:29); ORF-M (SEQ ID NO:30, encoding the protein set forth in SEQ ID NO:31); ORF-O (SEQ ID NO:32, encoding the protein set forth in SEQ ID NO:33); and ORF-Q (SEQ ID NO:34, encoding the protein set forth in SEQ ID NO:35).

[0040] In another aspect, the present invention provides isolated nucleic acid molecules that are at least 70% identical (such as at least 80% identical, or at least 90% identical, or at least 95% identical, or at least 99% identical) to a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

[0041] The present invention also provides isolated nucleic acid molecules that comprise a PDTC gene cluster as defined herein. The isolated nucleic acid molecules of the invention that comprise a PDTC gene cluster may optionally further comprise: a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO:7; a nucleic acid sequence that is at least 70% identical (such as at least 80% identical, or at least 90% identical, or at least 95% identical, or at least 99% identical) to the nucleic acid sequence set forth in SEQ ID NO:9; and a nucleic acid sequence that is at least 70% identical (such as at least 80% identical, or at least 90% identical, or at least 95% identical, or at least 99% identical) to the nucleic acid sequence set forth in SEQ ID NO:11. The present invention further provides isolated nucleic acid molecules that are at least 70% identical (such as at least 80% identical, or at least 90% identical, or at least 95% identical, or at least 99% identical) to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:13.

[0042] Nucleic acid molecules of the present invention can be isolated by a variety of cloning techniques known to those of ordinary skill in the art. For example, nucleic acid molecules having the nucleic acid sequences set forth herein (or portions thereof) can be used as hybridization probes utilizing, for example, the technique of hybridizing radiolabeled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes as set forth at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2^(nd) edition), Sambrook et al. eds., the cited pages of which are incorporated herein by reference. The hybridization probes may be labeled with appropriate reporter molecules. Exemplary means for producing specific hybridization probes include oligolabeling, nick translation, end-labelling or PCR amplification using a labeled nucleotide. Appropriate hybridization conditions can be readily calculated by one of ordinary skill in the art. For example, with respect to nucleic acid molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25° C. to 30° C. (for example, 10° C.) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987). Tm for nucleic acid molecules greater than about 100 bases can be calculated by the formula Tm=81.5+0.41% (G+C−log(Na⁺). With respect to nucleic acid molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5° to 10° C. below Tm. On average, the Tm of a nucleic acid molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) degrees centigrade.

[0043] Oligonucleotides for hybridization screening may be designed based on the DNA sequences of one or more of the nucleic acid molecules of the invention disclosed herein. Oligonucleotides for screening are typically at least 11 bases long and more usually at least 20 or 25 bases long. In one embodiment, the oligonucleotide is 20-30 bases long. Such an oligonucleotide may be synthesized in an automated fashion. To facilitate detection, the oligonucleotide may be conveniently labeled, generally at the 5′ end, with a reporter molecule, such as a radionuclide, (e.g., ³²P), enzymatic label, protein label, fluorescent label, or biotin. A library is generally plated as colonies or phage, depending upon the vector, and the recombinant DNA is transferred to nylon or nitrocellulose membranes.

[0044] Hybridization conditions are tailored to the length and GC content of the oligonucleotide. Oligonucleotides for hybridization are typically at least 11 bases long, generally less than 100 bases long, and preferably at least 15 bases long, such as at least 20 bases long, or at least 25 bases long, and preferably 20-70, 25-50, or 30-40 bases long. Washing is initially performed at the same conditions as hybridization. If the background is unacceptably high, washing temperature is increased a few degrees until background is acceptable.

[0045] Following denaturation, neutralization, and fixation of the DNA to the membrane, membranes are hybridized with labeled probe. Suitable hybridization conditions may be found in Sambrook et al., supra, Ausubel et al., supra, and furthermore hybridization solutions may contain additives such as tetramethylammonium chloride or other chaotropic reagents or hybotropic reagents (e.g., ammonium trichloroacetate; see for example, WO 98/13527) to increase specificity of hybridization.

[0046] Following hybridization, suitable detection methods reveal hybridizing colonies or phage that are then isolated and propagated. Candidate clones or amplified fragments may be verified as containing a desired nucleic acid sequence by any of various means. For example, the candidate clones may be hybridized with a second, non-overlapping probe or subjected to DNA sequence analysis.

[0047] Again, by way of example, nucleic acid molecules of the present invention can be isolated by the polymerase chain reaction (PCR) described in The Polymerase Chain Reaction (Mullis et al., eds., Birkhauser Boston (1994)), incorporated herein by reference. Template genomic DNA can be obtained from any bacterial species, such as from any Pseudomonas species. Additionally, nucleic acid molecules of the present invention can be synthesized in an automated fashion.

[0048] Nucleotide sequence variants of nucleic acid molecules of the present invention are useful provided that they encode a protein that retains the biological activity of the wild-type protein. Nucleotide sequence variants are nucleic acid molecules with some differences in their sequences as compared to the corresponding, native, i.e., naturally-occurring, nucleic acid molecules. Ordinarily, the variants will possess at least about 70% identity with the corresponding native sequences, and preferably, they will be at least about 80% identical to the corresponding, native sequences. The nucleic acid sequence variants falling within this invention possess substitutions, deletions, and/or insertions at certain positions. Sequence variants may be used to attain desired enhanced or reduced activity, or altered temporal and spatial patterns of activity. Such sequence variants can be generated by a variety of art-recognized techniques.

[0049] By way of non-limiting example, the two primer system utilized in the Transformer Site-Directed Mutagenesis kit from Clontech (Palo Alto, Calif.), may be employed for introducing site-directed mutations into nucleic acid molecules of the present invention. Following denaturation of the target plasmid in this system, two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site. Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli. This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids. The tight linkage of the two mutations and the subsequent linearization of unmutated plasmids results in high mutation efficiency and allows minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than prepare each positional mutant separately, a set of “designed degenerate” oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously. Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be fully sequenced or restricted and analyzed by electrophoresis on Mutation Detection Enhancement gel (J. T. Baker, Sanford, Me.) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control).

[0050] In other aspects, the present invention provides vectors that include one or more nucleic acid molecules of the invention, and host cells (including bacterial and plant cells) that include one or more vectors of the invention. Vectors useful for introducing the nucleic acid molecules of the invention into plant cells can be based, for example, on the Ti plasmid of Agrobacterium tumefaciens. The construction of suitable vectors containing nucleic acid molecules of the invention and optional elements, such as regulatory sequences and phenotypic selection genes utilize standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). The nucleic acid molecules and vectors of the invention may be prepared by manipulating the various elements to place them in proper orientation. Thus, adapters or linkers may be employed to join the DNA fragments. Other manipulations may be performed to provide for convenient restriction sites, removal of restriction sites or superfluous DNA. These manipulations can be performed by art-recognized methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0051] Prokaryotes may be used as host cells for routine genetic manipulation and/or construction of the nucleic acid molecules and vectors of the invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E. coli, such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are generally transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells. Prokaryote transformation techniques are set forth in Dower, in Genetic Engineering, Principles and Methods, 12:275-296, Plenum Publishing Corp. (1990); and Hanahan et al., Meth. Enzymol., 204:63 (1991).

[0052] In other aspects, the present invention provides cells (such as plant cells and bacterial cells) that include one or more vectors of the invention. Vectors of the invention can be introduced into plant cells using techniques well known to those skilled in the art. These methods include, but are not limited to, (1) direct DNA uptake, such as particle bombardment or electroporation (see, Klein et al., Nature 327:70-73 (1987); U.S. Pat. No. 4,945,050), and (2) Agrobacterium-mediated transformation (see, e.g., U.S. Pat. Ser. Nos: 6,051,757; 5,731,179; 4,693,976; 4,940,838; 5,464,763; and 5,149,645). Within the cell, the transgenic sequences may be incorporated within the chromosome. The skilled artisan will recognize that different independent insertion events may result in different levels and patterns of gene expression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., MGG 218:78-86 (1989)), and thus that multiple events may have to be screened in order to obtain lines displaying the desired expression level and pattern.

[0053] Transgenic plants can be obtained, for example, by transferring vectors that include a selectable marker gene, e.g., the kan gene encoding resistance to kanamycin, into Agrobacterium tumifaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature, 303:179-181 (1983) and culturing the Agrobacterium cells with leaf slices, or other tissues or cells, of the plant to be transformed as described by An et al., Plant Physiology, 81:301-305 (1986). Transformation of cultured plant host cells is normally accomplished through Agrobacterium tumifaciens.

[0054] Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, for example, kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.

[0055] In addition to the methods described above, several methods are known in the art for transferring cloned DNA into a wide variety of plant species, including gymnosperms, angiosperms, monocots and dicots (see, e.g., Glick and Thompson, eds., Methods in Plant Molecular Biology, CRC Press, Boca Raton, Fla. (1993), incorporated by reference herein). Representative examples include electroporation-facilitated DNA uptake by protoplasts in which an electrical pulse transiently permeabilizes cell membranes, permitting the uptake of a variety of biological molecules, including recombinant DNA (Rhodes et al., Science, 240:204-207 (1988)); treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant Molecular Biology, 13:151-161 (1989)); and bombardment of cells with DNA-laden microprojectiles which are propelled by explosive force or compressed gas to penetrate the cell wall (Klein et al., Plant Physiol. 91:440-444 (1989) and Boynton et al., Science, 240(4858):1534-1538 (1988)). A method that has been applied to Rye plants (Secale cereale) is to directly inject plasmid DNA, including a selectable marker gene, into developing floral tillers (de la Pena et al., Nature 325:274-276 (1987)). Further, plant viruses can be used as vectors to transfer genes to plant cells. Examples of plant viruses that can be used as vectors to transform plants include the Cauliflower Mosaic Virus (Brisson et al., Nature 310:511-514 (1984); Other useful techniques include: site-specific recombination using the Cre-lox system (see, U.S. Pat. Ser. No. 5,635,381); and insertion into a target sequence by homologous recombination (see, U.S. Pat. Ser. No. 5,501,967). Additionally, plant transformation strategies and techniques are reviewed in Birch, R. G., Ann Rev Plant Phys Plant Mol Biol, 48:297 (1997); Forester et al., Exp. Agric., 33:15-33 (1997). The aforementioned publications disclosing plant transformation techniques are incorporated herein by reference, and minor variations make these technologies applicable to a broad range of plant species.

[0056] Positive selection markers may also be utilized to identify plant cells that include a vector of the invention. For example, U.S. Pat. Ser. Nos. 5,994,629, 5,767,378, and 5,599,670 describe the use of a beta-glucuronidase transgene and application of cytokinin-glucuronide for selection, and use of mannophosphatase or phosphmanno-isomerase transgene and application of mannose for selection.

[0057] The cells which have been transformed may be grown into plants by a variety of art-recognized means. See, for example, McConnick et al., Plant Cell Reports 5:81-84 (1986). These plants may then be grown, and either selfed or crossed with a different plant strain, and the resulting homozygotes or hybrids having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.

[0058] The following are representative plant species that are suitable for genetic manipulation in accordance with the present invention. The citations are to representative publications disclosing genetic transformation protocols that can be used to genetically transform the listed plant species. Rice (Alam, M. F. et al., Plant Cell Rep. 18:572-575 (1999)); maize (U.S. Pat. Ser. Nos. 5,177,010 and 5,981,840); wheat (Ortiz, J. P. A., et al., Plant Cell Rep. 15:877-881 (1996)); tomato (U.S. Pat. Ser. No. 5,159,135); potato (Kumar, A., et al., Plant J. 9:821-829 (1996)); cassava (Li, H-Q., et al., Nat. Biotechnology 14:736-740 (1996)); lettuce (Michelmore, R., et al., Plant Cell Rep 6:439-442 (1987)); tobacco (Horsch, R. B., et al., Science 227:1229-1231 (1985)); cotton (U.S. Pat. Ser. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Ser. Nos. 5,187,073 and 6,020,539); peppermint (X. Niu et al., Plant Cell Rep. 1765-171 (1998)); citrus plants (Pena, L. et al., Plant Sci. 104: 183-191 (1995)); caraway (F. A. Krens, et al., Plant Cell Rep., 17:39-43 (1997)); banana (U.S. Pat. Ser. No. 5,792,935; soybean (U.S. Pat. Ser. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. Ser. No. 5,952,543); poplar (U.S. Pat. Ser. No. 4,795,855); monocots in general (U.S. Pat. Ser. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Ser. Nos. 5,188,958; 5,463,174 and 5,750,871); and cereals (U.S. Pat. Ser. No. 6,074,877).

[0059] The vectors used in this inventions are introduced into plant cells by any suitable technique. If the marker gene is a selectable gene, only those cells that have incorporated the DNA package survive under selection with the appropriate phytotoxic agent. Progeny from the transformed plants may be tested to ensure that the DNA package has been successfully integrated into the plant genome. The presence of the stably integrated elements into the transformed parent plants may be ascertained, for example, by southern hybridization techniques or PCR analysis, known in the art.

[0060] The nucleic acid molecules and vectors of the invention can be introduced into any desired microbial species, such as, but not limited to: Bacillus, Deinococcus, Thermus, Caulobacter, Methylobacterium, Alcaligenes, Burkholderia, Thiobacillus, Shingomonas, Flavobacterium, Achromatium, Acinetobacter, Actinobacillus, Aeromonas, Azotobacteriaceae, Beggiotoaceae (Beggiatoa), Chromateaceae, Collwellia, Coxiella, Ectothiorhodspira, Enterobacteriaceae, Legionellaceae, Methylococcaeeae, Moraxellaceae, Pateurellaceae, Pseudomonas, Shewanella, Thiomicrospira, Thiothrix, Vibrionaceae and Micrococcus radioduranis. An exemplary method for introducing the nucleic acid molecules of the invention into a bacterium by conjugation is set forth in Example 2 herein.

[0061] In another aspect, the present invention provides isolated proteins encoded by the nucleic acid molecules of the invention. By way of representative example, the present invention provides isolated proteins that are at least 70% identical to one or more of the proteins consisting of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. The proteins of the invention can be isolated, for example, by constructing a vector that includes a nucleic acid molecule that encodes the desired protein. The nucleic acid molecule is typically under the control of a regulatory element (e.g., an inducible promoter) that directs translation of the nucleic acid molecule. The vector is introduced into a cell (such as a bacterial cell, including bacteria of the genus Pseudomonas) and the expressed protein is purified therefrom. Representative examples of art-recognized techniques for purifying, or partially purifying expressed proteins of the invention from cells include ion-exchange chromatography, exclusion (gel permeation) chromatography, hydrophobic interaction chromatography, reversed-phase chromatography and immobilized metal affinity chromatography.

[0062] Hydrophobic interaction chromatography and reversed-phase chromatography are two separation methods based on the interactions between the hydrophobic moieties of a sample and an insoluble, immobilized hydrophobic group present on the chromatography matrix. In hydrophobic interaction chromatography the matrix is hydrophilic and is substituted with short-chain phenyl or alkyl nonpolar groups. The mobile phase is usually an aqueous salt solution. In reversed phase chromatography the matrix is silica that has been substituted with n-alkyl chains, usually C₄-C₁₈. The matrix is less polar than the mobile phase. The mobile phase is usually a mixture of water and a less polar organic modifier.

[0063] Separations on hydrophobic interaction chromatography matrices are usually done in aqueous salt solutions, which generally are nondenaturing conditions. Samples are loaded onto the matrix in a high-salt buffer and elution is by a descending salt gradient. Separations on reversed-phase media are usually done in mixtures of aqueous and organic solvents, which are often denaturing conditions. In the case of protein and/or peptide purification, hydrophobic interaction chromatography depends on surface hydrophobic groups and is carried out under conditions which maintain the integrity of the protein molecule. Reversed-phase chromatography depends on the native hydrophobicity of the protein and is carried out under conditions which expose nearly all hydrophobic groups to the matrix, i.e., denaturing conditions.

[0064] Ion-exchange chromatography is designed specifically for the separation of ionic or ionizable compounds. The stationary phase (column matrix material) carries ionizable functional groups, fixed by covalent bonding to the stationary phase. These fixed charges carry a counterion of opposite sign. This counterion is not fixed and can be displaced. Ion-exchange chromatography is named on the basis of the sign of the displaceable charges. Thus, in anion ion-exchange chromatography the fixed charges are positive and in cation ion-exchange chromatography the fixed charges are negative.

[0065] Retention of a molecule on an ion-exchange chromatography column involves an electrostatic interaction between the fixed charges and those of the molecule, binding involves replacement of the nonfixed ions by the molecule. Elution, in turn, involves displacement of the molecule from the fixed charges by a new counterion with a greater affinity for the fixed charges than the molecule, and which then becomes the new, nonfixed ion.

[0066] The ability of counterions (salts) to displace molecules bound to fixed charges is a function of the difference in affinities between the fixed charges and the nonfixed charges of both the molecule and the salt. Affinities in turn are affected by several variables, including the magnitude of the net charge (depends on pH) of the molecule and the concentration and type of salt used for displacement.

[0067] Solid-phase packings used in ion-exchange chromatography include cellulose, dextrans, agarose, and polystyrene. The exchange groups used include DEAE (diethylaminoethyl), a weak base, that will have a net positive charge when ionized and will therefore bind and exchange anions; and CM (carboxymethyl), a weak acid, with a negative charge when ionized that will bind and exchange cations. Another form of weak anion exchanger contains the PEI (polyethyleneimine) functional group. This material, most usually found on thin layer sheets, is useful for binding proteins at pH values above their pI. The polystyrene matrix can be obtained with quaternary amine functional groups for strong base anion exchange or with sulfonic acid functional groups for strong acid cation exchange. Intermediate and weak ion-exchange materials are also available. Ion-exchange chromatography need not be performed using a column, and can be performed as batch ion-exchange chromatography with the slurry of the stationary phase in a vessel such as a beaker.

[0068] Gel filtration is performed using porous beads as the chromatographic support. A column constructed from such beads will have two measurable liquid volumes, the external volume, consisting of the liquid between the beads, and the internal volume, consisting of the liquid within the pores of the beads. Large molecules will equilibrate only with the external volume while small molecules will equilibrate with both the external and internal volumes. A mixture of molecules (such as peptides) is applied in a discrete volume or zone at the top of a gel filtration column and allowed to flow through the column. The large molecules are excluded from the internal volume and therefore emerge first from the column while the smaller molecules, which can access the internal volume, emerge later. The volume of a conventional matrix used for protein purification is typically 30 to 100 times the volume of the sample to be fractionated. The absorbance of the column effluent can be continuously monitored at a desired wavelength using a flow monitor.

[0069] High Performance Liquid Chromatography (HPLC) is an advancement in both the operational theory and fabrication of traditional chromatographic systems. HPLC systems for the separation of biological macromolecules vary from the traditional column chromatographic systems in three ways; (1) the column packing materials are of much greater mechanical strength, (2) the particle size of the column packing materials has been decreased 5- to 10-fold to enhance efficiency of separation, and (3) the columns are operated at 10-60 times higher mobile-phase velocity. Thus, by way of non-limiting example, HPLC can utilize exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography and immobilized metal affinity chromatography and analytical and preparative mode.

[0070] In other aspects, the present invention provides methods for reducing the amount of a metal in a substrate (such as soil or water). Representative examples of metals that can be removed in accordance with the methods of this aspect of the invention include: iron, copper, chromium, cobalt, nickel, zinc, cadmium, lead, plutonium, arsenic, gold, titanium, tin, palladium, neodymium, gallium, bismuth, scandium and manganese. One embodiment of the methods of the invention for reducing the amount of a metal in a substrate comprises the steps of: (a) introducing into a substrate, comprising a metal ion species, a plant comprising roots and a PDTC gene cluster, the plant possessing a mechanism for transporting the metal ion species into the roots; and (b) expressing the PDTC gene cluster in the plant roots to form PDTC under conditions that enable the plant to remove an amount of the metal ion species from the substrate that is greater than the amount of the metal ion species that the plant would remove in the absence of expression of the PDTC gene cluster in the plant roots.

[0071] Plants comprising a PDTC gene cluster can be generated by any art-recognized technique for stably introducing DNA molecules (such as a nucleic acid molecule including a PDTC gene cluster) into plant cells and regenerating plants from the modified plant cells. The nucleic acid molecule comprising the PDTC gene cluster is typically utilized as part of a vector that is capable of replicating and being stably maintained within plant cells. Representative vectors and examples of genetic manipulation techniques for introducing a PDTC gene cluster into plant cells are set forth supra in connection with the description of plant host cells that include a vector of the invention. Representative nucleic acid molecules of the invention that encode a PDTC gene cluster are also described herein.

[0072] The PDTC gene cluster is expressed in the roots of plants (and, optionally, may also be expressed in other plant organs and tissues) under the control of one or more regulatory elements active in at least some cells (preferably all cells) of the plant roots. Representative examples of regulatory elements useful in this aspect of the invention include the Cauliflower Mosaic Virus 35S promoter. PDTC at or near the surface of the plant roots binds metal ions present in the substrate (such as soil or water contaminated with metal ions) and thereby enhances the availability of the metal ions to the endogenous (or genetically engineered) metal ion transport system of the plant, thereby enabling the plant to remove an amount of one or more metal ion species from the substrate that is greater than the amount of the metal ion species that the plant would remove in the absence of expression of the PDTC gene cluster in the plant roots.

[0073] Another embodiment of the methods of the invention for reducing the amount of a metal in a substrate comprises the steps of: (a) introducing into the rhizosphere of a plant at least one bacterial species that comprises a PDTC gene cluster, the rhizosphere comprising a metal ion species and the plant possessing a mechanism that transports the metal ion from the rhizosphere into the plant roots; (b) culturing the plant and the at least one bacterial species in the substrate for a time and under conditions that enable the bacterial species to synthesize PDTC and thereby increase availability of the metal ion to the plant roots so that the plant removes an amount of the metal ion from the substrate that is greater than the amount of the metal ion that the plant would remove if the bacterial species expressing PDTC was not present in the rhizosphere.

[0074] The at least one bacterial microorganism that comprises a PDTC gene cluster can be constructed by introducing into a desired bacterial species one or more nucleic acid molecules that include a PDTC gene cluster. The nucleic acid molecule(s) comprising the PDTC gene cluster is typically incorporated into a vector that is capable of replicating and being stably maintained within the target microorganism(s). Vectors useful in this aspect of the invention typically include (a) a nucleic acid molecule comprising the PDTC gene cluster, (b) a selectable marker gene (typically an antibiotic-resistance gene, such as a gene that confers resistance to ampicillin or kanamycin) for identification and recovery of microorganisms that incorporate the PDTC gene cluster, and (c) appropriate regulatory elements to control expression of the PDTC biosynthetic genes and selectable marker gene(s). Representative examples of inducible promoters useful for regulating the expression of the PDTC biosynthetic genes and/or the selectable marker gene(s) are Ptac (the activity of which is induced by IPTG) and Pm (the activity of which is induced by m-toluic acid). (See, e.g., Marques et al., Mol. Microbiol. 9(5):923-929 (1993); Yap et al., J. Bacteriol, 176(9):2603-2610 (1994); and Cebolla et al., Appl. Environ. Microbiol., 61(2):660-668 (1995)).

[0075] The nucleic acid molecules of the invention can be introduced into any desired microbial species, such as, but not limited to: Bacillus, Deinococcus, Termus, Caulobacter, Methylobacterium, Alcaligenes, Burkholderia, Thiobacillus, Shingomonas, Flavobacterium, Achromatium, Acinetobacter, Actinobacillus, Aeromonas, Azotobacteriaceae, Beggiotoaceae (Beggiatoa), Chromateaceae, Collwellia, Coxiella, Ectothiorhodspira, Enterobacteriaceae, Legionellaceae, Methzylococcaeeae, Moraxellaceae, Pateurellaceae, Pseudomonas, Shewanella, Thiomicrospira, Thiothrix, Vibrionaceae and Micrococcus radiodurans. An exemplary method for introducing the nucleic acid molecules of the invention into a bacterium is set forth in Example 2 herein.

[0076] PDTC (synthesized by the bacteria that have been engineered to include a PDTC gene cluster) at or near the surface of the plant roots binds metal ions present in the substrate (such as soil or water contaminated with metal ions) and thereby enhances the availability of the metal ions to the plant's metal ion transport system, enabling the plant to remove an amount of one or more metal ion species from the substrate that is greater than the amount of the metal ion species that the plant would remove if the bacterial microorganism expressing PDTC was not present.

[0077] In a related aspect, the present invention provides methods for immobilizing metal ions in a substrate. In this aspect of the invention, one or more species of microorganism, comprising a PDTC gene cluster, are introduced into a substrate containing one or more metal ions under conditions that enable expression of the PDTC gene cluster to form PDTC which is released into the substrate. The PDTC binds the metal ions thereby reducing the extent of movement (such as by diffusion) of the metal ions through the substrate. Methods of this aspect of the invention are useful, for example, for treating soil contaminated with metal ions, thereby reducing the ability of the metal ions to move throughout the soil.

[0078] The complex formed between PDTC and Cu(II) ions is capable of degrading carbon tetrachloride to yield carbon dioxide and hydrogen chloride. Thus, the present invention provides methods for degrading carbon tetrachloride, such as carbon tetrachloride contaminating soil or water.

[0079] One embodiment of the methods of the invention for degrading carbon tetrachloride includes the steps of: (a) introducing into a substrate a plant comprising roots and a PDTC gene cluster, the substrate further comprising Cu(II) ions and carbon tetrachloride that are introduced into the substrate, before, after or simultaneously with the introduction of the plant into the substrate; and (b) expressing the PDTC gene cluster in the plant roots under conditions that enable the plant to synthesize PDTC and release the PDTC into the substrate, thereby chemically degrading the carbon tetrachloride by the action of a complex formed between the PDTC and the Cu(II) ions.

[0080] Another embodiment of the methods of the invention for degrading carbon tetrachloride includes the steps of: (a) introducing into a substrate, comprising Cu(II) ions and carbon tetrachloride, at least one bacterial species that comprises a PDTC gene cluster under conditions that enable expression of the PDTC gene cluster to form PDTC; and (b) release of the PDTC into the substrate thereby chemically degrading the carbon tetrachloride by the action of a complex formed between the PDTC and the Cu(II) ions. The Cu(II) ions are introduced into the substrate before, after, or simultaneously with the bacterial species.

[0081] Another embodiment of the methods of the invention for degrading carbon tetrachloride in a substrate includes the steps of: (a) introducing into a substrate, comprising carbon tetrachloride and Cu(II) ions, a nucleic acid molecule comprising a PDTC gene cluster comprising a plurality of PDTC biosynthetic genes, each of the PDTC biosynthetic genes being operably linked to at least one regulatory element that directs their expression within a microorganism; (b) uptake of the introduced nucleic acid molecule by a microorganism; (c) expression of the PDTC gene cluster within the microorganism to form PDTC; and (d) release of the PDTC into the substrate thereby chemically degrading the carbon tetrachloride by the action of a complex formed between the PDTC and the Cu(II) ions. In this embodiment of the methods of the invention, the nucleic acid molecule comprising the PDTC gene cluster can be introduced into soil by attachment (such as by absorption or adsorption) of the nucleic acid molecule to clay particles. Clay useful in this aspect of the invention includes colloidal clays such as bentonite and montmorillonite. Colloidal sand particles (such as colloidal sand produced by grinding pure silica) is also useful in this aspect of the invention. Typically, the particle size of the clay or sand is less than 0.002 mm.

[0082] The nucleic acid molecule comprising the PDTC gene cluster can be attached to the clay or sand particles by any art-recognized method, such as the methods described in Lorenz, M. G., et al., J. Gen. Microbiol. 134:107-112 (1988); Sikorski, J., et al., Microbiology 144:569-576 (1998); Lorenz, M. G., and Wackernagel, Arch. Microbiol. 154:380-385 (1990); Khanna, M., and Stotzky, G, Appl. Environ. Microbiol. 58:1930-1939 (1992); Nielsen, K. M., et al., Appl. Environ. Microbiol. 63:1945-1952 (1997); Romanowski, G., et al., Mol. Ecol. 2:171-181 (1993); Romanowski, G., et al., Appl. Environ. Microbiol. 57:1057-1061 (1991). Once introduced into the soil, the nucleic acid molecule is taken up by one or more species of microorganism (typically bacteria) by natural processes.

[0083] The nucleic acid molecule comprising the PDTC gene cluster is typically utilized as part of a vector that is capable of replicating and being stably maintained within the target microorganism(s). Vectors useful in this aspect of the invention typically include (a) a nucleic acid molecule comprising the PDTC gene cluster, (b) a selectable marker gene (typically an antibiotic-resistance gene, such as a gene that confers resistance to ampicillin or kanamycin) for identification and recovery of microorganisms that incorporate the PDTC gene cluster, and (c) appropriate regulatory elements to control expression of the PDTC biosynthetic genes and selectable marker gene(s). Representative examples of inducible promoters useful for regulating the expression of the PDTC biosynthetic genes and/or the selectable marker gene(s) are Ptac (the activity of which is induced by IPTG) and Pm (the activity of which is induced by m-toluic acid).

[0084] In another aspect, the present invention provides compositions (such as the DNA molecule consisting of the sequence set forth in SEQ ID NO:13) and methods for transferring genes between bacterial species. Thus, for example, the DNA molecule consisting of the sequence set forth in SEQ ID NO:13 can be used to transfer genes included therein between species of Pseudomonas.

[0085] In other aspects of the invention, PDTC and certain of its metal complexes can be used to degrade carbon tetrachloride and for removing metals from substrates, such as soil and water. These advantageous properties render PDTC and certain of its metal complexes useful in environmental remediation methods including, for example, phytoremediation, bioaccumulation, water purification, waste water purification, solution mining mobilization, immobilization, detoxification, redox state modifier, and modification of metal ion reactivity. The chemical structure of PDTC is illustrated in FIG. 2A. Modes of PDTC binding to certain metals are illustrated in FIGS. 2B-2E, which show chemical structures of representative PDTC metal complexes.

[0086] As noted above, the present invention provides compositions and methods for degrading carbon tetrachloride. The compositions and methods of the invention useful in carbon tetrachloride degradation include PDTC and certain of its metal complexes. Representative PDTC complexes that are useful in carbon tetrachloride degradation include the PDTC copper (II) complex and PDTC cobalt complex.

[0087] In general, PDTC copper (II) complex can be a carbon tetrachloride contaminated substrate, such as soil or water, to facilitate carbon tetrachloride degradation. The amount of PDTC copper complex applied to the substrate will depend on the substrate and the amount of carbon tetrachloride present in the substrate. The PDTC copper complex can be applied as a solution (such as a saturated solution in water) or as a solid. The PDTC copper complex can also be used, for example, in an organic solvent such as methanol or dimethylformamide.

[0088] The complex formed between PDTC and Cu(II) ions is capable of degrading carbon tetrachloride to yield carbon dioxide and hydrogen chloride. Thus, the present invention provides methods for degrading carbon tetrachloride, such as carbon tetrachloride contaminating soil or water.

[0089] In one embodiment, the method for degrading carbon tetrachloride includes the steps of: (a) contacting PDTC copper (II) complex with a substrate contaminated with carbon tetrachloride; and (b) chemically degrading the carbon tetrachloride by the action of a complex.

[0090] Carbon tetrachloride degradation by PDTC is described in Example 3. As described in Example 3, carbon tetrachloride dechlorination by PDTC requires copper and is inhibited by cobalt, but not by iron or nickel. PDTC reacts stoichiometrically, rather than catalytically, without added reducing equivalents. With added reductants, an increased turnover was seen, along with increased chloroform production.

[0091] Carbon tetrachloride (CCl₄) and its dechlorination products have been thoroughly studied, primarily to understand their toxic and carcinogenic effects in mammals, and their environmental fate. In the liver, CCl₄ is transformed by cytochrome P₄₅₀ through reductive mechanisms. Reactive species such as trichloromethyl radical and dichlorocarbene, thought to be responsible for cell damage due to CCl₄ exposure, are produced. Microbial transformations of CCl₄ have also been studied to evaluate the potential for engineered bioremediation or natural attenuation of CCl₄-contaminated environments. The only biochemical agents from microbial sources that have been studied for their CCl₄ dechlorination activity are tetrapyrrole-type cofactors, including cobalamius, porphyrins, and factor F₄₃₀. These cofactors also mediate the transformation of CCl₄ through a reductive mechanism.

[0092] The data on dehalogenation mechanisms involving transition metal cofactors, which include product analyses and spectroscopic studies, indicate an initial one-electron reduction to give radical species. The dominant fate of the resulting carbon-centered radicals in these systems is another one-electron reduction by the bulk reductant, which is present to regenerate the active form of the cofactor. This net two-electron reduction yields hydrogenolytic products (i.e., replacement of one chlorine atom by one hydrogen atom). Other products resulting from the net two-electron reduction of CCl₄ are carbon monoxide and formate, which arise through hydrolysis of dichlorocarbene.

[0093] Another distinct type of CCl₄ dechlorination activity has been described in cultures of iron-limited Pseudomonas stutzeri strain KC. This activity is characterized by an extensive hydrolysis that gives CO₂ as a major product, as well as uncharacterized non-volatile material and low or undetectable levels of chloroform. Chloroform is not dechlorinated by this organism, indicating that CCl₄ is degraded via a novel pathway that avoids the accumulation of less-chlorinated products. Thiophosgene (CSCl₂) was identified as an intermediate of the net hydrolysis in our earlier studies. Quantitative data obtained using trapping agents demonstrated that the pathway involving thiophosgene accounts for most of the CCl₄ transformation observed in strain KC cultures under anoxic conditions. Oxygen substitution at the carbon atom of CCl₄ was also observed in the form of carbonyl-containing products, which were found to increase when O₂ was present. If trichloromethyl radical were involved, oxygen substitution could be attributed to a phosgene intermediate, likely to occur in the presence of O₂, but not under anoxic conditions. Another intermediate explaining carbonyl substitution under anoxic conditions and arising from thiophosgene hydrolysis is carbonyl sulfide (COS), which is also likely to be trapped by the nucleophiles used. An abiotic CCl₄ transformation which affects the substitution of sulfur for chlorine occurs in mineral/sulfide mixtures. The data in those studies suggested a radical substitution mechanism, initiated by one-electron reduction of CCl₄ at a metal center and followed by reaction of trichloromethyl radical with one of a variety of sulfur species that may have been present.

[0094] Pyridine-2,6-bis(thiocarboxylic acid) is an extracellular agent responsible for CCl₄ dechlorination activity in strain KC. PDTC has been identified as a metal-chelating agent from iron-limited cultures of a strain of Pseudomonas putida. The occurrence of two thiocarboxylic groups in PDTC and its ability to coordinate transition metals suggested a potential mechanism for reaction with CCl₄ analogous to that proposed for the mineral/sulfide system; specifically, reduction at the metal center to produce trichloromethyl radical, and condensation of this radical with one of the sulfur atoms of PDTC. The addition of certain transition metals to cultures of P. stutzeri strain KC has been found to exert profound effects on CCl₄ transformation activity. Fe(II) and Fe(III) prevented CCl₄ transformation when present initially in culture media at 10-100 μM, but not when added to cultures already showing this activity. Co(II) was found to inhibit CCl₄ transformation in low micromolar concentrations, and to inhibit growth at higher concentrations. CuCl₂ stimulated CCl₄ transformation activity at very low concentrations (5 nM), and had an inhibitory effect on growth of bacteria at higher concentrations. Inhibition of dechlorination may be due to the formation of inactive metal-containing complexes in preference to the active dechlorinating species. This scenario did not explain the data obtained from iron supplementation experiments. An alternative hypothesis was that inhibition or stimulation of dechlorination activity might be due to repression or induction of PDTC biosynthesis, respectively. Data obtained from transposon mutants derived from strain KC indicate a repression of genes necessary for CCl₄ transformation in response to iron supplementation. The hypotheses for direct chemical effects of transition metals and physiological effects are not mutually exclusive; however, previous experiments have not allowed the clear resolution of these effects.

[0095] Another unexplained phenomenon was observed in studies attempting to characterize the extracellular dechlorination agent. These studies showed that either bacterial cells or a chemical reductant were required in order to observe dechlorination in culture supernatants. The rationale for the use of a chemical reductant was that the responsible agent might be a redox catalyst that could couple oxidation of a chemical reductant or cell-derived reducing equivalent to reductive dechlorination of CCl₄. This rationale was weakened upon determining that the agent itself contained sulfur and was the likely source of the sulfur atom transferred to the CCl₄ carbon atom, thus indicating a stoichiometric rather than catalytic reaction with respect to PDTC. The requirement for reductant then became somewhat puzzling. Questions regarding the effects of transition metals on PDTC dechlorination activity, and its chemical requirements, have important ramifications for the in situ use of PDTC in biological or chemical reactive treatments. The identification of PDTC as the active agent and the availability of chemically-synthesized PDTC has made experiments possible that can resolve chemical from biological effects and allow product analyses without the complications arising from the presence of bacterial cells.

[0096] Contamination of soils and water with metals is a problem in many areas of the world. Applying existing cleanup methods to these sites is expensive. Phytoextraction is a remediation strategy to remove metals from the soil by virtue of metal uptake into plant tissues and subsequent removal of that plant material. Development of a system employing plants to remove metals from the soil could substantially reduce the cost of remediating metal-contaminated sites. Augmenting current and future phytoremediation systems with microbial partners possessing exploitable traits such as excretion of specific metal-chelating molecules may be a way to speed up and broaden the application of phytoextraction techniques to existing contamination problems.

[0097] Certain cultivars of Indian mustard (Brassica juncea) have been found to accumulate significant levels of Cr, Cd, Ni, Zn, Cu, and Pb (58-, 52-, 31-, 17-, 7-, and 1.7-fold, respectively) in harvestable tissues when grown in metal-amended soil, as measured by phytoextraction coefficient (PC). The phytoextraction coefficient for a specific metal is determined by dividing the μg of metal per gram of dried harvestable plant tissue by the μg metal per gram of dry soil ((μg of metal/g DW plant tissue)/(μg of metal/g DW soil)).

[0098] Amending soils with metal-chelating molecules has been shown to be effective in substantially increasing Pb uptake and its subsequent translocation to shoot tissues of corn, pea, ragweed, goldenrod, and sunflower. Increased uptake of Cd, Zn, Mn, Cu, Fe, Al, and Ni has also been observed when bushbeans were grown in the presence of synthetic chelators. Metal-chelating molecules are thought to act by increasing the solubility of metals and thereby increasing the bioavailability of the metals for uptake and translocation by plants. Se and Hg uptake into the tissues of saltmarsh bulrush and rabbitfoot grass has been shown to be higher when naturally occurring rhizosphere bacteria are present than under axenic conditions. This mode of action of this enhancement was not elucidated by the researchers, but may be due to siderophore production by rhizosphere bacteria. Siderophores are iron-chelating molecules secreted by bacteria.

[0099] PDTC is a small molecule secreted by certain pseudomonads when subjected to iron-limiting conditions which forms complexes with many metals. PDTC is a candidate for phytoextraction enhancement studies for several reasons. Most siderophores chelate iron(III) with hydroxymate or catecholate ligands, but PDTC binds metals with various combinations of its pair of (thiocarboxylate) ligands combined with a single secondary amine (see FIGS. 2A-2E). Iron is chelated by most siderophores using 6 coordinating ligands. However, while most siderophores utilize 3 bi-dentate ligands, PDTC uses 2 tri-dentate ligands. At 197 daltons, PDTC is smaller than most bacterially produced siderophores, which have molecular weights in the range of 500 to 1000 daltons. Since PDTC is a small and relatively simple molecule it can be produced economically, either by bacterial cultures or synthetically. Its small size also increases the diffusion rate of the free molecule and its chelates. PDTC has an affinity to a wide range of metals including Au, Cd, Co, Cr, Cu, Fe, Mn, Nd, Ni, Pb, Pd, Sc, and Zn. As noted above, PDTC can also be produced in situ by several pseudomonads possibly including those which preferentially inhabit the rhizosphere of any given plant species. Because of its unique properties, PDTC can be used to expand the spectrum of metal contamination problems that can be addressed by phytoextraction.

[0100] Because PDTC is a metal chelator and forms strong metal complexes, PDTC can be used to bind and remove metal ions from a substrate. Thus, in another aspect of the invention, compositions and methods for chelating metals ions are described. These compositions and methods include PDTC and can be used in environmental remediation programs including, for example, phytoremediation, bioaccumulation, water purification, and solution mining, among others. In these methods, PDTC-containing compositions can be directly applied to metal-containing substrates, such as soil or water systems.

[0101] The usefulness of chelators in bioremediation efforts often depends on their ability to bind hazardous metal ions. Since most known microbial chelators have a high specificity toward iron, their value as bioremediation agents is severely diminished due to the relatively high availability of iron compounds in many natural settings, allowing iron to outcompete other metals for binding to the chelator. Advantageously, PDTC complexes are formed with numerous transition and heavy metals, lanthanides and actinides. PDTC is soluble in water or supercritical fluids, and can function in these solvents as an extractant of metals from most or all biological tissues and many environmental matrixes including, for example, groundwater, soils, and ores. PDTC complexes can be formed with transition metals, lanthanide metals, actinide metals, heavy metals, and radionuclides. Representative metals that could be complexed with PDTC include Cu, Sc, Ti, Mn, Ni, Cu, Fe, Zn, Cr, Co, As, Au, Pd, Cd, Pb, Hg, Nd, Tc, Sr, tin, gallium and bismuth, among others.

[0102] The present invention provides methods for immobilizing metal ions in a substrate. In this aspect of the invention, PDTC is contacted with a substrate containing one or more metal ions. The PDTC binds the metal ions thereby reducing the extent of movement (such as by diffusion) of the metal ions through the substrate. Methods of this aspect of the invention are useful, for example, for treating soil contaminated with metal ions, thereby reducing the ability of the metal ions to move throughout the soil. In one embodiment, the method is a phytoremediation method. In such a method, a plant having an ability to take up the PDTC metal complex can be introduced into the substrate (i.e., soil) such that the metal ion, through its complexation with PDTC, ultimately resides in the plant. The plant containing the metal ion can then be removed from the substrate thereby remediating the substrate.

[0103] The stability of various PDTC-metal complexes was determined by experiments on potentiometric and spectrophotometric studies of PDTC with several metals. The stability constants and relative binding strengths for PDTC and several of the physiologically important metals that it binds are described in Example 4.

[0104] To summarize, PDTC metal chelating was studied by potentiometric and spectrophotometric techniques as described in Example 4. The first two stepwise stability constants (log K) for successive proton addition to PDTC were found to be 5.48 and 2.58. The third stepwise stability protonation constant was estimated to be 1.3. The stability constant for cobalt(III), copper(II), nickel(II), and iron(III) were determined spectrophotometrically by competition experiments. The stability constants (log K) are, respectively, 33.93 (Co), 33.28 (Ni), and 33.36 (Fe). The constant for ferric PDTC was determined by competitive experiments with the hydroxide ion. Constants for cobalt(III), copper(II), and nickel(II) were determined by metal-metal competition with respect to the stability constant of ferric PDTC.

[0105] The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.

EXAMPLES Example 1

[0106] This example describes the cloning and characterization of a fragment (SEQ ID NO:13) of the Pseudomonas stutzeri genome that encodes enzymes involved in the biosynthesis of PDTC.

[0107] Characterization of strain CTN1, a spontaneous mutant defective in CCl₄ transformation: While studying CCl₄ transformation by P. stutzeri strain KC, the inventors noted a loss of this activity in a working stock of this organism. The loss of activity could not be attributed to medium composition; in fact, when frozen stocks of strain KC were revived and tested on the same medium (DRM), the normal activity was seen (not shown). It was subsequently determined that no detectable PDTC was produced by the inactive culture (CTN1; Table 1). TABLE 1 PDTC production by strain KC and complementations of strain [PDTC] Host strain Plasmid (μmoles/mg protein) P. stutzeri strain KC none 28.4 ± 4.6 P. stutzeri CTN1 none N.D. P. stutzeri CTN1 pM22 2.55 ± 0.33 P. stutzeri CTN1 pM36 N.D. P. stutzeri CTN1 pO9 N.D. P. stutzeri CTN1 pT31 49.8 ± 2.9 P. stutzeri CTN1 pJS18 53.0 ± 1.1 P. stutzeri CTN1 pJS20 45.1 ± 11.3 P. stutzeri CTN1 pJS22 54.1 ± 19.8 P. stutzeri CTN1 pJS27 57.7 ± 3.3 P. stutzeri CTN1 pJS29 53.2 ± 7.6 P. stutzeri CTN1 pJS34 N.D. P. stutzeri CTN1 pJS40  3.5 ± 0.5 P. stutzeri CTN1 pJS42 56.1 ± 7.5 P. stutzeri CTN1 pJS43 40.3 ± 0.6 P. stutzeri CTN1 pJS52 55.6 ± 3.3 P. stutzeri CTN1 pJS55 35.1 ± 0.4 P. stutzeri CTN1 pJS59 N.D. P. stutzeri CTN1 pJS60 N.D. P. stutzeri CTN1 pJS63 55.9 ± 16.9 P. stutzeri CTN1 pJS68 N.D. P. stutzeri CTN1 pT-phoA1 N.D.

[0108] The inventors have since observed this loss of CCl₄ transformation activity on several occasions (not shown). Further correlation of PDTC biosynthesis and CCl₄ dehalogenation was demonstrated by P. putida DSM3601, the culture from which PDTC was originally identified (Ockels, W., Römer, A., and Budzikiewicz, H., Tetrahedron Lett. 3341-3342 (1978)), which shows CCl₄ transformation activity in an iron-limited medium (not shown). The loss of PDTC production (Pdt⁻phenotype) during maintenance of strain KC led the inventors to suspect a defect in a genetic locus required for PDTC biosynthesis. However, the rapid CCl₄ transformation activity of strain KC (≧1 μg CCl₄ ml⁻¹ day⁻¹; Lewis, T. A., and Crawford, R. L., “Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC” Appl. Environ. Microbiol. 59:1635-1641 (1993)) was the only known phenotype distinguishing strain KC from other strains of P. stutzeri and therefore, the inventors could not easily rule out contamination. Consequently, to ensure that the inactive culture was that of a clonal variant of strain KC and not a random contaminant, several tests were performed. Biolog™ substrate utilization plates reproducibly identified the culture as P. stutzeri (data not shown). A partial sequence of the 16S ribosomal RNA subunit genes of strain KC (GenBank accession No. AF063219) and the Pdt⁻variant showed no differences in an approximately 500 base-pair segment. Genomic fingerprinting with rare-cutting restriction enzymes and pulsed-field agarose gel electrophoresis (PFGE) has been shown to readily resolve strains of P. stutzeri (Ginard et al. “Genome organization of Pseudomonas stutzeri and resulting taxonomic and evolutionary considerations” Int. J. Syst. Bact. 47:132-143 (1997)). Comparing strain KC and the Pdt⁻ variant by this technique clearly showed their clonal relatedness. The Pdt⁻ variant was then designated as strain CTN1.

[0109] Identification of a chromosomal deletion in strain CTN1: The appearance of a variant in PDTC biosynthesis without intentional selection suggested that a mutation characterized by a relatively high frequency had occurred. Such mutations could include plasmid loss, transposon or phage excision, as well as homologous (legitimate) recombination leading to deletion or inversion of chromosomal DNA. PFGE of undigested DNA did not indicate the presence of plasmids in strain KC or CTN1 (not shown), though this type of analysis did not rule out the existence of very large plasmids that did not enter the gel. The SpeI restriction patterns of the two strains differed discernibly only with respect to one band, of approximately 148 kb. This difference could be explained by a number of possible scenarios including point mutation, inversion, or deletion. To see if a gross rearrangement of the chromosome had occurred, we used extremely rare-cutting restriction enzymes. The intron-encoded I-CeuI, whose recognition site is found in bacterial rrl genes, gives four large fragments of P. stutzeri chromosomes, designated in descending size as CeA through CeD (Ginard et al. “Genome organization of Pseudomonas stutzeri and resulting taxonomic and evolutionary considerations” Int. J. Syst. Bact. 47:132-143 (1997)). Analyses of several pulsed-field gels revealed a dimorphism in the CeB fragment corresponding to a loss of 172±54 kb in CTN1. Dimorphism of the third largest PacI fragment (PaC) and loss of the PaE and PaF fragments corresponded to a net loss of 171±11 kb in strain CTN1. To locate the deletion on a circular map of the strain KC chromosome, Southern blotting analysis was performed. PFGE separations of single, as well as double, I-CeuI and PacI digests of chromosomal DNAs of strains KC and CTN1 were probed with a set of cloned loci, or PFGE-purified PaC, PaD, PaE, PaF fragments. This allowed construction of a crude map and localization of the deletion.

[0110] Cloning of pdt genes of strain KC: Chromosomal rearrangements may be a common event in pseudomonads and it was possible that the deletion present in CTN1 was unrelated to the Pdt⁻ phenotype. A more detailed study of the DNA deleted in strain CTN1 was undertaken to determine if it was involved in determining the Pdt phenotype. To obtain cloned DNA from the deleted region we used the 148 kb Spe1 fragment of strain KC as a probe to screen a strain KC genomic library. Several cosmids were identified and transferred into CTN1 by conjugation. The transconjugants were then tested for complementation of the Pdt⁻ phenotype by assaying CCl₄ transformation. One cosmid, pT31, was found to complement the Pdt⁻ phenotype, restoring PDTC production (Table 2) and CCl₄ transformation activity to CTN1. A map of the pT31 insert (SEQ ID NO:13), showing the locations of open reading frames, is set forth in FIG. 1. TABLE 2 PDTC production by non-PDTC-producing heterologous hosts of P. stutzeri strain KC pdt genes. [PDTC]in medium (μmoles/mg protein) Host organism Cosmid pRK311 Cosmid pT31 P. stutzeri CTN1 N.D.  49.8 ± 2.9 P. putida mt-2 N.D. 100.5 ± 11.1 P. fluorescens F113 N.D.  71.5 ± 30.9 P. aeruginosa PAO1 N.D.  34.8 ± 8.8 P. stutzeri ATCC 17588 N.D.  5.3 ± 2.7 R. meliloti N.D. N.D. E. coli ATCC 25922 N.D. N.D.

[0111] This complemented mutant probably produced higher amounts of PDCT and greater CCl₄ transformation activity than strain KC because of the increased copy number of the cosmid-borne insert DNA. pT31 was found to contain an insert of 25,746 bp of DNA (SEQ ID NO:13) that was colinear with the strain KC chromosome. pT31 was used in cosmid walking experiments to align other clones tested for complementation and to localize the necessary region (FIG. 1). pM22, another cosmid with an approximately 25 kb genomic DNA insert, restored only 10% of wild-type level of PDTC production and a reduced level of CCl₄ transformation activity (FIG. 1, Table 1). Southern blotting with the entire pT31 molecule as a probe showed that sequences included on this construct were not present in CTN1, indicating that it lay entirely within the deletion. pT31 also did not hybridize with genomic DNAs from any of three culture collection strains of P. stutzeri (ATCC 11607, ATCC 14405, and ATCC 17588) (not shown). As this region was shown by deletion and complementation to be required for PDTC biosynthesis, it was designated the pdt locus.

[0112] The ability of pT31 to confer PDTC production and rapid CCl₄ transformation activity was not limited to strain CTN1 as host, but was also seen with other strains of P. stutzeri, and with strains of other Pseudomonas species that normally show no such activity (Table 2). Control pseudomonad strains containing only the cosmid vector, E. coli, or Rizobium meliloti harboring pT31, showed a Pdt⁻ phenotype (Table 2).

[0113] Sequence analysis of the pdt locus: the insert (SEQ ID NO:13) of pT31 was sequenced and analyzed in order to identify potential open reading frames (GenBank accession no. AF196567). Comparison of this sequence data with that of an 8.27 kb EcoRI fragment from strain KC described by Sepulveda-Torres et al. (GenBank accession No. AF149851) showed that these two EcoRI fragments were the same. Sepulveda-Torres et al. obtained the sequence after cloning from mutants lacking CCl₄ transformation activity as a result of mutagenesis by a specialized transposon (Sepulveda-Torres et al. “Generation and initial characterization of Pseudomonas stutzeri KC mutants with impaired ability to degrade carbon tetrachloride” Arch. Microbiol, 171:424-429 (1999)). To assess the function of various portions of the pT31 insert (SEQ ID NO:13) in Pdt complementation, pT31 was mutagenized using mini-Tn5 constructs (mini-Tn5 lacZ1, mini-Tn5 phoA (DeLorenzo et al. “Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria” J. Bacteriol. 172:6568-6572 (1990))). After mapping these insertions, their effect on PDTC production by strain CTN1 was measured (FIG. 1, Table 1). Although some open reading frames were not disrupted by any of the mapped insertions, an assessment of the extent of pT31 DNA required for complementation of the Pdt⁻ phenotype was afforded (FIG. 1).

[0114] Insertions that significantly influenced complementation activity were found in ORF-F (SEQ ID NO:14), ORF-J (SEQ ID NO:16), ORF-K (SEQ ID NO:1), and ORF-P (SEQ ID NO:5) (see FIG. 1). Homology searches showed that these ORFs encode proteins with significant similarities with MoeB/MoeZ sulfurylases, an AMP ligase, a receptor protein, and a methyltransferase, respectively (see Table 3). TABLE 3 Putative open reading frames (RFs) encoded within the pT31 insert (SEQ ID NO: 13). ORF/ SEQ ID Start Ending NO: Base Base AA kDa pl Homology A/30 1 360 >119 >13.8 NA Similar to acyl-CoA synthase from Mycobacterium bovis (AAB52538); BLOSUM62 expect = 5 × 10⁻¹⁰, 36% identities and 53% positives over 121 aa overlap. B/22 158 1099 313 34.2 10.39 Probable thioesterase. Similar to gramicidin S biosynthesis GRST protein from Brevibacillus brevis (P14686); BLOSUM62 expect = 3 × 10⁻²³, 35% identities and 49% positives over 188 aa overlap. C/7 4066 3353 235 26.2 9.88 Probable transcriptional activator. Similar to XylS/AraC transcriptional activator from Salmonella typhimurium (3094022); BLOSUM62 expect = 2 × 10⁻⁹, 31% identities and 50% positives over 107 aa overlap. Has 25% identities and 50% positives to 40 aa AraC bacterial regulatory protein family protein signature (PROSITE PS00041). D/24 4486 5052 188 20.1 8.64 No significant homology. E/26 5187 6302 371 40.3 11.13 No significant homology. Has 11 predicted transmembrane domains. F/14 6475 7650 391 43.7 4.88 Possible sulfurylase. Similar to MoeZ from Mycobacterium tuberculosis (CAB08310); BLOSUM62 expect = 10⁻¹²², 57% identities and 71% positives over 388 aa overlap. Has a single predicted transmembrane domain located between residues 43 to 65. G/9 7666 8076 136 15.6 6.68 Similar to hypothetical 16.5 kd protein RV1334 precursor from Mycobacterium tuberculosis (Q10645); BLOSUM62 expect = 8 × 10⁻³⁰, 47% identities and 67% positives over 134 aa overlap. H/11 8139 8411 90 9.7 5.61 Similar to hypothetical protein from Streptomyces coelicolor A3 (CAB50992); BLOSUM62 expect = 6 × 10⁻²¹, 48% identities and 74% positives over 90 aa overlap. Lower homologies to MoaD proteins. I/18 8446 10332 628 67.5 6.98 Similar to putative racemase from Rhodococcus sp. (CAB55821); BLOSUM62 expect = 2 × 10⁻²³, 30% identities and 44% positives over 285 aa overlap. J/16 10278 12023 581 62.8 5.51 Probable AMP ligase. Similar to 2,3- dihydroxybenzoate-AMP ligase from Bacillus subtilis (P40871); BLOSUM62 expect = 3 × 10⁻⁵⁰ , 28% identities and 45% positives over 529 aa overlap. Has AMP binding motif between residues 211-222. K/1 11974 14037 687 75.3 5.93 Probable receptor precursor. Similar to FyuA precursor from Escherichia coli (CAA84488); BLOSUM62 expect = 2 × 10⁻²⁹, 25% identities and 40% positives over 586 aa overlap. Has 25 aa signal peptide located at amino terminus. Has TonB-dependent receptor protein signature (PROSITE PS00430). L/28 14069 16300 743 83.3 7.50 No significant homologies. M/30 16716 19010 764 83.0 6.09 Probable aminotransferase. Similar to SC6A5.18 from Streptomyces coelicolor (CAB39702); BLOSUM62 expect = 4 × 10⁻⁷⁰, 34% identities and 50% positives over 467 aa overlap. N/3 19073 20251 392 40.6 10.72 Probable ABC transporter. Similar to YycB from Bacillus subtillis (CAB16085); BLOSUM62 expect = 5 × 10⁻⁹, 24% identities and 41% positives over 325 aa overlap. O/32 23041 21500 513 57.4 8.78 Probable acyl-CoA dehydrogenase. Similar to DR0922 from Deinococcus radiodurans; BLOSUM62 expect = 1 × 10⁻⁷⁰, 38% identities and 58% positives over 386 aa overlap. P/5 23969 22914 351 38.2 5.82 Probable methyltransferase. Similar to hydroxyneurosporene methyltransferase (CrtF) from Rhodobacter sphaeroides CRTF_RHOSH); BLOSUM62 expect = 1 × 10⁻⁹, 30% identities and 40% positives over 204 aa overlap. Has 67% identities to S-adenosylmethionine- dependent methyltransferase motifs (Kagan and Clarke, 1994). Q/34 25588 25746 >53 >6.4 NA Similar to predicted coding region AF1178 (AAB90082) from Archaeoglobus fulgidus; BLOSUM62 expect = 2 × 10⁻⁴, 45% identities and 74% positives over 36 aa overlap.

[0115] Genbank accession numbers are in parentheses when provided.

[0116] The hypothetical MoeZ protein derives its name from its very high similarity to several molybdopterin synthase sulfarylase (MoeB) proteins. MoeB, along with the MoaD/MoaE heterodimer, molybopterin synthase, act to sulfurylate precursor Z of the molybdenum cofactor biosynthetic pathway (Rajagopalan, “Biosynthesis of the molybdenum cofactor.” In Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. Neidhardt (ed). Washington D.C.: ASM Press, pp. 674-679 (1996)). The only mini-transposon insertion into ORF-F (SEQ ID NO:14) obtained in this study was pT-phoA1, a mini-Tn5 phoA insertion. This mini-transposon construct contains an alkaline phosphatase gene that is functionally expressed only when an in-frame translational fusion is created within an extracytoplasmic domain of a protein. A single predicted transmembrane segment is located at residues 43-65 of the 390 amino acid ORF-F product (SEQ ID NO:15). The phoA gene of pT-phoA1 was mapped to the carboxyterminal half of the protein (FIG. 1). In addition to the homology shared by the translated product of ORF-F (SEQ ID NO:15) and M. tuberculosis MoeZ, ORF-F (SEQ ID NO:14) also has nucleic acid homology to moeZ of M. tuberculosis (GenBank accession No. Z95120). The BLOSUM62 expect value for this homology was 9×10⁻¹⁵ with 80% identity over 194 bases (not shown). The AMP ligase, EntE, participates in the synthesis of the siderophore enterochelin by 2,3-dihydroxybenzoate adenylation and thioester coupling to pantetheinyl-EntB (Adams and Schumann “Cloning and mapping of the Bacillus subtilis locus homologous to Escherichia coli ent genes” Gene 133:119-121 (1993); Gehring et al. “Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate” Biochemistry 36:8495-8503 (1997)). Both the mini-Tn5 insertions in ORF-J (SEQ ID NO:16) (pJS60 and pJS34) abolished PDTC production (Table 1). FyuA is an outer membrane protein involved in the uptake of ferric yersiniabactin in a TonB- and proton motive force-dependent fashion (Moeck and Coulton “TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport” Mol. Microbiol. 28:675-681 (1998); Pelludat et al. “The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation” J. Bacteriol. 180:538-546 (1998); Rakin et al. “The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function” Mol. Microbiol. 13:253-263 (1994)). A variable effect of transposon disruption of ORF-K (SEQ ID NO:1) was seen, since one insertion (pJS59) resulted in a loss of PDTC production while the other (pJS52) did not (Table 1). A disruption of ORF-P (SEQ ID NO:5), the methyltransferase homolog (pJS40, Table 1, FIG. 1), resulted in low but detectable PDTC production. Deletion of ORF-P (SEQ ID NO:5) and a portion of ORF-O (SEQ ID NO:32) (pM22, FIG. 1) resulted in reduced CCl₄ transformation activity and low levels of PDTC production. ORF-P (SEQ ID NO:5) is transcribed in the opposite orientation to the other 3 ORFs shown experimentally to be required for normal PDTC production. The deletion introduced by construction of pJS68 removed a portion of ORF-N (SEQ ID NO:3) in addition to sequences truncated in pM22. This resulted in a loss of detectable PDTC production (FIG. 1, Table 1).

[0117] ORFs that were disrupted by mini-transposon insertions without significant effect on measured PDTC concentrations were ORF-C (SEQ ID NO:7), ORF-D (SEQ ID NO:24), ORF-E (SEQ ID NO:26), ORF-L (SEQ ID NO:28), and ORF-M (SEQ ID NO:30). ORFs that were not mutagenized by mini-transposon insertions were ORF-A (SEQ ID NO:20), ORF-B (SEQ ID NO:22), ORF-G (SEQ ID NO:9), ORF-H (SEQ ID NO:11), ORF-I (SEQ ID NO:18), ORF-N (SEQ ID NO:3), ORF-O (SEQ ID NO:32) and ORF-Q (SEQ ID NO:34). The coding regions of ORF-A (SEQ ID NO:20) and ORF-Q (SEQ ID NO:34) are incomplete in pT31 (Table 3, FIG. 1).

[0118] While not wishing to be bound by theory, the foregoing presumed functions of the proteins encoded by the open reading frames within SEQ ID NO:13, and the results of the foregoing transposon insertion experiments, suggest a biosynthetic pathway for PDTC as well as auxiliary functions for export and uptake. The (thiocarboxylate) groups may be generated by sulfurylation of a carboxylic acid precursor (by the ORF-F/MoeZ homolog and possibly ORF-G (SEQ ID NO:9) and H (SEQ ID NO:11) as accessory subunits) that has been activated by adenylation (by the ORF-J (SEQ ID NO:16) product/Ent E homolog). A novel aspect of this proposed synthesis pathway is that the sulfur atom remains bonded to the carbonyl carbon, whereas nonribosomal peptide synthase reactions using the thiotemplate mechanism (e.g., Ent B/G) effect substitution alpha to the carbonyl carbon. The finding that an ORF-F (SEQ ID NO:15)-phoA fusion produces active alkaline phosphatase and therefore is localized to a membrane suggests that PDTC biosynthesis occurs in proximity to the cytoplasmic membrane. Little is known regarding siderophore export but others have found evidence of membrane association of siderophore biosynthetic enzymes (Hantash and Earhart “Membrane association of the Escherichia coli enterobactin synthase proteins EntB/G, EntE, and EntF” J. Bacteriol. 182:1768-1773 (2000)). A potential exporter was identified in the ORF-N gene (SEQ ID NO:3), which shows homology with ABC transporters. Other evidence for ABC transporter involvement in siderophore export was found in the exochelin system of Mycobacterium smegmatis (Zhu et al. “Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes” Mol. Microbiol. 29:629-639 (1998)). Receptor-mediated uptake of PDTC-transition metal complexes is suggested by the ORF-K gene (SEQ ID NO:1). The apparent involvement of this gene product in PDTC biosynthesis or regulation is novel among siderophore regulatory networks. Other membrane receptors have been identified with autoregulatory functions (Venturi et al. “Gene regulation of siderophore-mediated iron acquisition in Pseudomonas: not only the Fur repressor” Mol. Microbiol. 17:603-610 (1995)), but no effects on siderophore biosynthetic genes were noted.

[0119] In view of the foregoing, it is therefore a further aspect of the present invention to provide isolated nucleic acid molecules that are at least 70% identical (such as at least 80% identical, or at least 90% identical, or at least 95% identical, or at least 99% identical) to any one of the nucleic acid molecules consisting of the nucleic acid sequences set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 and SEQ ID NO:34.

[0120] It is a further aspect of the present invention to provide isolated protein molecules that are at least 70% identical (such as at least 80% identical, or at least 90% identical, or at least 95% identical, or at least 99% identical) to any one of the protein molecules consisting of the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33 and SEQ ID NO:35.

[0121] It is yet a further aspect of the present invention to provide isolated nucleic acid molecules that hybridize to a nucleic acid molecule consisting of a nucleic acid sequence selected from the group of nucleic acid sequences consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ED NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 and SEQ ID NO:34, under conditions of 1×SSC at 60° C. for 20 minutes, or to the complement of any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 and SEQ ID NO:34, under conditions of 1×SSC at 60° C. for 20 minutes.

Example 2

[0122] This example sets forth the experimental materials and techniques used to generate the data set forth in Example 1.

[0123] Bacterial strains and culture conditions: Bacteria used in this study are listed in Table 4. TABLE 4 Bacteria and plasmids used in this study Strain/Plasmid GENOTYPE SOURCE/REFERENCE E. coli DH5α F⁻end A1 hsdR17(r_(K) ⁻m_(K) ⁺) supE44 thi-1 Gibco BRL recA1 gyrA relA1 Φ80dlacZΔM15 Δ(lacZYA-argF)U169 E. coli S17-1 Sm^(r) chromosomal RP4 integrant S. Minnich, University of (mob⁺) Idaho (Simon et al., 1983) E. coli SM10(λ Km^(r), thi-1, thr, leu, tonA, lacY, supE, K. Timmis, GBF, pir) recA::RP4-2-Tc::Mu, λpir Braunschweig E. coli Top-10 F⁻, mcrAΔ(mrr-hsd/RMS-mcrBC), Invitrogen Corp. Φ80 lacZΔM15, ΔlacX74deoR, recA1, araD139Δ(ara-leu)7697, ga/U, galK, rpsL, (Str^(r)) endA, nupG E. coli HB101 (Sm^(r)) recA, thi, pro, leu, hsdR-M P. stutzeri KC Wild type, aquifer isolate C. Criddle, Stanford University (Criddle et al., 1990) P. stutzeri CTN1 Spontaneous derivative of strain KC This study P. stutzeri 17588 Wild type, clinical isolate (type strain) ATCC* P. stutzeri 14405 Wild type, marine isolate ATCC (ZoBell) P. stutzeri 11607 Wild type, clinical isolate ATCC P. fluorescens Wild type soil isolate F. O'Gara, University of F113 Cork, Ireland P. aeruginosa Wild-type N. Schiller, University of PAO1 California, Riverside P. putida Wild type DSMZ** DSM3601 P. putida mt-2 Wild-type toluene degrader University of Idaho culture collection, (Williams and Murray, 1974) R. meliloti 102f34 Wild-type G. Ditta, University of California, San Diego Plasmid Ap^(r) Stratagene, La Jolla, Calif. pBluescriptSK+ Plasmid pRK311 Tc^(r) broad host range cosmid (mob⁻ tra⁺) M. Kahn, Washington State University (Ditta et al., 1985) Plasmid pT31 Pdt⁺ pRK311 carrying ˜25.5 kb insert of P. stutzeri strain KC genomic DNA, this study Plasmid pUT-Km Amp^(r), Km^(r), delivery plasmid for mini- K. Timmis mini-Tn5 lacZ1 Tn5 lacZ1 Plasmid pUT-Km Amp^(r), Km^(r), delivery plasmid for mini- K. Timmis mini-Tn5 phoA Tn5 phoA Plasmid pRK2073 Conjugation plasmid G. Ditta

[0124] For routine growth of E. coli, LB medium was used (Sambrook et al. Molecular cloning: A laboratory manual Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989)). For testing CCl₄ transformation activity, M9 minimal medium with glucose (Sambrook et al. Molecular cloning: A laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989)) and supplemented with 0.1 mM L-leucine and 5 nM CuCl₂ was used. Antibiotics used were from Sigma (St. Louis, Mo.) at the following concentrations: tetracycline (Tc, 7.5 μg/mL), ampicillin (Ap, 50 μg/mL), kanamycin (Km, 75 μg/ml), spectinomycin (Sp, 25 μg/ml), and nalidixic acid (Nal, 15 μg/ml). Substrate utilization tests employed Biolog™ plates (Biolog Inc., Hayward, Calif.). P. stutzeri were maintained on tryptic soy agar. Isolation streaks used for maintenance of strain KC contained <300 isolated colonies. A single colony was selected at random for transfer to fresh medium at approximately monthly intervals for this purpose. Stocks were initiated from frozen (−80° C., 15% glycerol) aliquots semi-annually to annually to avoid selection of strains evolved on laboratory medium. CCl₄ transformation was assessed using HM-acetate medium (Lewis and Crawford “Physiological factors affecting carbon tetrachloride dehalogenation by denitrifying bacterium Pseudomonas sp. strain KC” Appl. Environ. Microbiol. 59:1635-1641 (1993)) or DRM, which contained the following (per liter): K₂HPO₄, 6 g; sodium acetate, 2 g; sodium nitrate, 0.5 g; ammonium chloride, 1 g; adjusted to pH 7.8 and autoclaved. After cooling, 1 mL of 1M MgSO₄, 10 mL of Ca(NO₃)₂, and 50 μL of 0.1 mM CuCl₂ were added from sterile stock solutions. P. putida was grown on minimal succinate medium (SM) which contained: K₂HPO₄, 6 g; KH₂PO₄, 3 g; (NH₄)₂SO₄, 1 g; MgSO₄.7H₂O, 0.2 g; succinic acid, 4 g; pH was adjusted to 7.6 with NaOH (Meyer and Abdallah, 1978). Cultures (100 mL) were inoculated from aerobically-grown overnight cultures (1% v/v) and grown in 160 mL serum bottles stoppered with Teflon mininert valves (Pierce, Rockford, Ill.). CCl₄ was added from methanolic stock solutions. Cultures were incubated inverted, with rotary shaking at 150 rpm in a 25° C. incubator. Growth was monitored by protein assay using the bicinchoninic acid method (Pierce) performed on 1 mL samples either precipitated with 1 M trichloroacetic acid and digested at 100° C. for 30 min. in 1 M NaOH or treated with 100 μL toluene to burst the cells.

[0125] Analytical techniques: CCl₄ concentrations were determined by headspace gas chromatography with an electron capture detector as described previously (Lewis and Crawford “Physiological factors affecting carbon tetrachloride dehalogenation by denitrifying bacterium Pseudomonas sp. strain KC” Appl. Environ. Microbiol. 59:1635-1641 (1993)). Samples (0.1 mL) were taken with a 1 mL gas tight syringe (Hamilton, Reno, Nev.) and placed in 10 mL headspace autosampler vials. PDTC was determined spectrophotometrically with a Hewlett Packard 8453 diode array spectrophotometer (Budzikiewicz et al. “Weitere aus dem Kulturmedium von Pseudomonas putida isolierte Pyridinderivate—Genuine Metaboliten oder Artefackte?” Z. Naturforsch 38b:516-520 (1983)).

[0126] Recombinant DNA techniques: A genomic library of strain KC was constructed using DNA partially digested with Sau3A and size fractionated on a 10-40% sucrose density gradient (Sambrook et al. Molecular cloning: A laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989)). Size-fractionated DNA (>approx. 20 kb) was ligated into pRK311 that had been treated with BamHI and shrimp alkaline phosphatase (Gibco). Ligation products were packaged into phage particles using Gigapack II XL extracts (Stratagene). These were used to infect E. coli DH5α, for titer determination and insert efficiency on X-Gal/IPTG, and E. coli S17-1 as the library host. The average insert size was determined to be 28 kb among 10 random picks. One thousand colonies were picked for storage in 15% glycerol and used to prepare colony lifts on nylon membranes (MagnaLift, Micron Separations Inc., Westboro, Mass.,). Cosmids were transferred into pseudomonads or other strains of E. coli by conjugation, performed by the patch mating technique (De Feyter and Gabriel “Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas cainpestris pv. Malvacearum” J. Bacteriol. 173:6421-6427 (1991)) on TSA plates (for pseudomonads) or LB plates (for E. coli). Matings were conducted for 3-6 h at 30° C. for pseudomonads or for 1-3 h at 37° C. for E. coli. Southern transfer of DNA restriction digests to nylon membranes (Hybond N, Amersham, UK) was done with a vacuum blotting apparatus (Hoefer Scientific TransVac, San Francisco, Calif.) or by capillary transfer (Sambrook et al. Molecular cloning: A laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989)). Probes for hybridization were synthesized using the RadPrime kit from Gibco and α³²P-dCTP (NEN, Boston, Mass.) or the NEBlot non-radioactive labeling kit (New England Biolabs, Beverly, Mass.). Pad and I-CeuI restriction endonucleases were from New England Biolabs; all others were from Gibco BRL.

[0127] 16S rDNA sequences were determined by PCR amplification from genomic DNA templates and primers designed from published sequences. The products were cloned into pBluescript KS+ and three individual clones from each product were sequenced using M13 forward and reverse primers and the Sequitherm EXCEL™ II Long-Read™ DNA sequencing kit (Epicentre Technologies Corp., Madison, Wis.) and a Li-Cor automated sequencer (Li-Cor Inc., Lincoln, Nebr.). The strain KC nosZ and recA genes and the chromosomal origin of replication (ori) of P. stutzeri strain KC were obtained by PCR amplification and cloning into TOPO TA (Invitrogen, Carlsbad, Calif.). PCR reactions used primers designed from published sequences (GenBank accession numbers M22628, L12684, and M30125 respectively). Identities of the cloned products were confirmed by sequencing and alignment searching using BLAST programs (Altschul et al. “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs” Nucleic Acids Res, 25:3389-3402 (1997)).

[0128] Pulsed field gel electromhoresis: Agarose-embedded DNAs from P. stutzeri were prepared and digested with restriction enzymes as described by Römling et al. (“Bacterial genome analysis by pulsed field gel electrophoresis techniques” In Advances in Electrophoresis. Chrambach, Dunn, and Radola (eds.), Wenheim, Germany: Wiley-VCH, pp. 335-406 (1994)). PacI digests were done by first allowing the enzyme to diffuse into the agarose at 4° C. for 3-16 h before incubation at 37° C. for 2 h. The digests were electrophoresed using a CHEF DRII or DRIII apparatus (Bio-Rad, Richmond, Calif.). Gels were run at 14° C. using molecular biology grade agarose (International Biotechnologies Inc., New Haven, Conn.) in 0.5×TBE (Sambrook et al. Molecular cloning: A laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989)) and an included angle of 120°. Agarose concentrations, voltages, and pulse lengths, optimized for particular experiments, are given in figure legends. Size standards used were Saccharomyces cerevisiae chromosomes, phage λ oligomers (New England Biolabs), λ HindIII digests (Sigma), and Hansenula wingei chromosomes (Bio-Rad).

[0129] DNA sequencing and sequence analysis: The insert of pT31 (SEQ ID NO:13) was sequenced by a combination of subcloning and primer walking. Dye termination reactions were processed on an ABI 377 sequencer (Perkin-Elmer, Foster City, Calif.). Assembly of sequence data was performed using OMIGA version 1.1 (Kramer 2000). From an initial set of potential open reading frames greater than 50 amino acids in length, 17 open reading frames were selected as potential coding regions by analysis of codon bias (Staden et al. “The Staden package” Meth. Mol. Biol. 2000 132:115-130 (2000)) (Table 3). Homology searches were conducted using BLASTP and BLASTN (Altschul et al. “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs” Nucleic Acids Res, 25:3389-3402 (1997)). For identification of protein domains and signatures, PROSITE and BLOCKS databases were searched using the ExPASy proteomics server and NCBI's Impala search engine, respectively (Appel et al. “A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server” Trends Biochem. 19:258-260 (1994); Henikoff and Henikoff “Protein family classification based on searching a database of blocks” Genomics 19:97-107 (1994)). Prediction of transmembrane regions of protein sequences was performed by the TMHMM program. Calculation of isoelectric points and molecular weights for putative protein sequences was performed by using the Wisconsin package (Butler “Sequence analysis using GCG” Meth. Biochem. Anal. 39:74-97 (1998)).

[0130] Transposon mutagenesis: Donor strain E. coli SM10 (pUT-Km mini-Tn5 lacZ1) or (pUT-Km mini-Tn5 phoA), helper strain E. coli HB101 (pRK2073), and recipient strain E. coli Top-10 (pT31) were grown overnight at 37° C. with antibiotic selection. 0.3 mL of each culture were combined and filtered through a 0.21 μm (pore size) filter (Supor®, Gelman Sciences, Ann Arbor, Mich.). Filters were placed on an LB plate and incubated for at least 8 hours at 37° C. Mating mixtures were then resuspended in sterile M9 medium (w/o carbon source). 0.1-0.5 mL of this suspension was spread on LB Km, Tc and grown at 37° C. overnight. The Ki^(r) Tc^(r) E. coli were resuspended by rinsing the plates with M9 medium. Tc^(R), Km^(R) plasmids in this suspension were recovered by “mating out” using DH5α as the recipient (3 h, 37° C.). DH5α (pT31-KmlacZ1) were amplified by plating on LB Km, Tc, Nal. For mini-Tn5 phoA, the Tc^(R), Km^(R) plasmids were mated out with CTN1 and plated on M9 citrate Km, Tc and BCIP (5-bromo-6-chloro-3-indolyl Phosphate, Sigma). Among several hundred colonies, one showed an intense blue color after 10 days. The cosmid present in this transconjugant (pT-phoA1) was transferred to DH5α for DNA preparation. The location of mini-Tn5 insertions was determined by single-digests with EcoRI and XhoI (lacZ1) or EcoRI and BamHI (phoA) and separation by electrophoresis through 0.8% agarose.

Example 3

[0131] Degradation of Carbon Tetrachloride by PDTC Metal Complexes

[0132] In this example, the degradation of carbon tetrachloride by PDTC metal complexes is described.

[0133] CCl₄ transformation assays. CCl₄ transformation assays were performed in 2 ml of 35 mM potassium phosphate buffer (KH₂PO₄/KOH, pH 7.7) prepared with glass-distilled deionized water and stored in an anaerobic chamber (Forma Scientific, atmosphere N₂:H₂:CO₂, 85:10:5) with 1 g of Chelex 100 chelating resin (Sigma Chemical Co., St. Louis, Mo.) per 100 ml. All glassware used for stock solutions or reactions was cleaned with aqua regia (conc. HCl:conc. HNO₃, 4:1, vol/vol). Reaction mixtures (2 ml) containing approximately 25 μM PDTC were prepared in the anaerobic chamber in 20-ml-headspace autosampler vials, and received a total of approximately 0.2 μmoles CCl₄. CCl₄ (Omnisolv, Merck) was added from a methanol stock solution (approx. 0.8%, vol/vol). Additions of CCl₄ were made with a 25-μl gas-tight syringe (Hamilton, Reno, Nev.) immediately before sealing with a Teflon-faced butyl rubber stopper (West Co., Phoenixville, Pa.) and aluminum crimp seal. Reactions were incubated at 25° C. in an inverted position in test tube racks for 72 hours unless otherwise noted.

[0134] Hydrogen sulfide was from Aldrich (Milwaukee, Wis.). Sodium sulfide (Na₂S×9H₂O) was from EM Science (Gibbstown, N.J.). Titanium(III) citrate was prepared in the anaerobic chamber from 20% Ti(III)Cl₃/HCl solution (Fisher), trisodium citrate, and sodium carbonate to give a final pH of 7.7 and a final concentration of 0.5M Ti. ¹³CCl₄ was from Cambridge Isotope Laboratories (Andover, Mass.) and ¹⁴CCl₄ was from DuPont NEN (Wilmington, Del.). CuCl₂, 99.999%, and FeCl₃×6H₂O, 98%, were from Aldrich. CoSO₄×7H₂O, 99%, was from Sigma. NiCl₂×6H₂O, 99.9999%, was from Fisher (Acros).

[0135] Bioassays. Pseudomonas stutzeri American Type Culture Collection strain 17588, known from previous work to have no significant CCl₄ transformation activity or PDTC biosynthetic capacity, was used in assays of CCl₄ transformation with PDTC. The organism was grown aerobically in a medium containing (per liter) dipotassium phosphate, 6 g; sodium acetate, 2 g; ammonium sulfate, 1 g; sodium nitrate, 0.5 g. The pH of the medium was adjusted to 7.7-7.9 with HCl before autoclaving, and, after cooling, calcium nitrate and magnesium sulfate were added from sterile stock solutions to final concentrations of 0.1 mM and 1 mM, respectively. Cultures were grown overnight at 30° C. Aliquots (1 ml) in 10 ml headspace autosampler vials were used in triplicate assays containing 50 μM PDTC and 30 nmoles CCl₄. These were incubated at 25° C., inverted, for 16 hours.

[0136] Synthesis of PDTC and its metal complexes. PDTC was synthesized by the method of Hildebrand et al., Phosphorus Sulfur, 16:361-364 (1983). Aqueous solutions were prepared by dissolving PDTCH₂ (5 mM) in anoxic 35 mM potassium phosphate buffer (pH 7.7), followed by filtration through 0.2-μ (pore size) membranes. The Cu—Cl and Cu—Br complexes were isolated as the tetrabutylammonium salts, as described for the synthesis of Pd—Br:PDTC complex (Espinet et al., Inorg. Chem. 33:2052-2055(1994)). Elemental analysis of the Cu—Br complex gave (theoretical in parentheses): C, 47.5% (47.37); H, 6.62% (6.74); N, 4.81% (4.80); S, 11.17% (11.00).

[0137] Analytical methods: Gas chromatography. For assays to determine the effectiveness of various metal ions in promoting CCl₄ dechlorination, determinations of CCl₄ were made by gas chromatography/mass spectroscopy (Thermoquest Trace GC/Trace MS, Thermo Separation Products, San Jose, Calif.). For optimal linearity of responses in the range of concentrations encountered, CCl₄ was detected by single ion monitoring (SIM) of the ion m/z 82; CHCl₃, m/z 83; CS₂, m/z 76 or 78. One milliliter injections were made by a headspace autosampler (HS2000) following a 10 minute conditioning cycle at 70° C. with shaking. The column used was a Supel-Q PLOT (30 m×0.32 mm, Supelco, Bellefonte, Pa.). Carbonyl sulfide (COS) and carbon disulfide were analyzed by headspace gas chromatography with a Hewlett Packard 6890 GC, a PoraPLOT Q column (Chrompack, Middelburg, The Netherlands) and a 5973 mass selective detector and integration of the total ion chromatograms (TIC—20-150 Daltons). Injections were made using a 7693 headspace autosampler. Vials were equilibrated to 70° C. for 10 min with shaking before injection to a 1-ml sample loop. The concentration of CCl₄ in bacterial cultures was measured using a Hewlett Packard (Avondale, Pa.) 5890 gas chromatograph equipped with an electron capture detector and a 19395 headspace autosampler. Standards were prepared by additions of analytes (CCl₄, CHCl₃, CS₂) from stock methanol solutions.

[0138] Radiotracer analysis. ¹⁴C-labeled CCl₄ was used to determine mass balances with a nitrogen-purging manifold, employing organic traps, base traps, and scintillation counting. This method gave 100±7% recovery of ¹⁴CO₂ from NaH¹⁴CO₃ and 93.2±1.1% for ¹⁴CCl₄.

[0139] Electrospray MS. The non-volatile species present in reaction mixtures was analyzed by negative or positive electrospray ionization tandem mass spectrometry (Quattro II, Micromass Ltd., UK). Concentrated reactions used for mass spectral identification of products were 2 mM Cu:PDTC and approximately 20 μl CCl₄ per ml of reaction volume in DMF:H₂O, 1:1. Samples were delivered into the source at a flow rate of 5 μL/min using a syringe pump (Harvard Apparatus, South Natick, Mass.). A potential of 2.5-3 kV was applied to the electrospray needle. The sample cone was kept at an average of 15 V. The counter electrode, skimmer, and RF lens potentials were tuned to maximize the ion beam for the given solvent. Resolution of the detector was 15,000 and source temperature was kept constant at 80° C. The instrument was calibrated using a polyethylene glycol solution. All spectra were an average of 10-15 scans.

[0140] Liquid chromatography. Chloride was measured using a Dionex 2010i ion chromatograph equipped with an AS4a column (Dionex, Sunnyvale, Calif.), Na₂CO₃/NaHCO₃ eluent at 2 ml/min, and suppressed conductivity detection.

[0141] Dipicolinic acid (pyridine-2,6-dicarboxylic acid) was measured on a Thermo Separations Products HPLC, SpectraSystem P2000 with an AS3000 autosampler and 10 μl injections. The column was a 4.6×250 mm 5μ Hypersil BDS C18. Analytes were eluted using 25 mM sodium phosphate pH 7.0, 5 mM tetrabutylammonium phosphate (TBAP) {A} and acetonitrile, 5 mM TBAP {B}. A gradient was generated by pumping 1 ml/min of 95% A and increasing to 65% B over 15 min. Detection was by UV absorption at 260 nm with spectral scanning using a UV6000LP photodiode array detector.

[0142] Electron Paramagnetic Resonance. EPR spectra were recorded at X-band frequencies using a Bruker ESP300E spectrometer (Bruker Instruments, Billerica, Mass.). Samples were loaded in a quartz flat cell (Wilmad Glass Co., Buena, N.J.) in an anaerobic chamber and sealed with Teflon stoppers and parafilm prior to transferring into the instrument sample cavity. Spectra were collected at room temperature as follows: microwave frequency 9.68 GHz, microwave power 20 mW, modulation frequency 100 KHz, modulation amplitude 1.0 G, conversion time 164 ms, time constant 328 ms, number of scans averaged 32, number of data points 4096, sweep width 75 gauss, sweep time per scan 671 s. The spin trap α-phenyl-tert-butyl nitrone (PBN, Sigma) was purified by vacuum sublimation prior to use. Trapping experiments were performed in the presence of 100 mM PBN.

[0143] The following summarizes the effect of transition metal ions on PDTC-mediated carbon tetrachloride transformation.

[0144] No added reductant. The metal ions tested included those known to have effects upon dechlorination by strain KC (Fe, Co, Cu) and/or known to form complexes with biological ligands that are active for catalytic dechlorination (Fe, Co, Ni). Without added transition metal, no significant CCl₄ transformation was seen (see Table 5 below). Copper was the only metal found to have a significant stimulatory effect on CCl₄ transformation. No inhibitory effects could be observed in these assays since the control (no metal addition) showed no significant transformation; however, CoSO₄ (13 μM) addition to PDTC prior to CuCl₂ addition (13 μM) led to no significant CCl₄ transformation. FeCl₃ addition (50 μM) prior to CuCl₂ addition did not prevent transformation.

[0145] Bacterial cells and chemical reducing agents. Conditions known to show PDTC-dependent CCl₄ transformation at trace copper concentrations were used to assess inhibitory effects of transition metals. These included suspensions of non-PDTC-producing bacterial cells, and chemical reductants. These conditions were not subject to inhibition of PDTC biosynthesis and therefore should detect only direct chemical effects. Variables introduced by the use of sulfide and Ti(III) as reductants are their respective redox potentials, which affect their ability to reduce the transition metals in complex with PDTC. For example Ti(III) (E_(o)′=−0.480V) has been shown to reduce Co(III) in cyanocobalamin to Co(I) (10, 14), but thiols such as dithiothreitol (E_(o)′=−0.332V) cannot reduce cyanocobalamin beyond Co(H) (11). Bacterial cells, sulfide, or Ti(III) citrate affected significant CCl₄ transformation in combination with PDTC without added transition metals (see Table 5 below). With bacterial cells or sodium sulfide, only cobalt addition resulted in an inhibition of CCl₄ transformation (see Table 5 below). With Ti(III) citrate, this effect was less pronounced and iron addition also resulted in decreased transformation.

[0146] The fact that cobalt effectively inhibited transformation and that copper stimulated it indicated the importance of metal ligation for dechlorination by PDTC. An explanation for these effects may be that copper was the only metal among those tested capable of promoting reaction between CCl₄ and PDTC. This requirement may have been met by very low concentrations of copper, but cobalt effectively excluded copper from the ligand. Atomic absorption spectroscopy determinations indicated that there was approximately 0.26 mmoles of copper per mole of PDTC, and therefore at least 6 nM copper was present in all reactions including PDTC. Time course experiments were performed to determine the effect of copper concentration on CCl₄ transformation, with and without reductants. The data are shown in FIG. 3. The kinetics of CCl₄ transformation were very rapid at 0.5 moles Cu/mole PDTC, and slower at the lower copper concentrations. Rate constants could not be determined with the limited data, but by simple observation, the extent of transformation at a given time point was dependent on copper concentration. When sulfide was present this effect was evident over an 8-hour time course with additions of only 75 nM CuCl₂. In contrast, when no reductant was added 188 nM CuCl₂ did not affect significant transformation over a 42-hour time period (FIG. 3). A differential response to copper imparted by the reductant was clearly evident in these data. This effect explains CCl₄ transformation by PDTC without added copper as due to the presence of copper contamination.

[0147] Stoichiometry of PDTC-dependent CCl₄ transformation and quantitation of hydrogenolysis products. The data described above were useful in identifying which metals had direct effects on dechlorination by PDTC, but did not indicate which conditions were most representative of dechlorination by strain KC. This determination required a more detailed description of the transformation products. Catalytic dechlorination agents such as corrinoids or hematin show a large proportion of hydrogenolytic products in the presence of excess reductant. The CCl₄ transformation seen with strain KC is characterized by very few hydrogenolytic products, but significant sulfur substitution. Chloroform and carbon disulfide were measured in the experiments of Table 5. Those data are given, along with CCl₄ turnover calculations, in Table 6. A stoichiometry of approximately two moles of CCl₄ per mole of PDTC was obtained when CuCl₂ was added without added reducing agents (see Table 5 below). These conditions also resulted in low amounts of chloroform and a significant amount of carbon disulfide. Sulfide addition led to much higher turnover of CCl₄ per mole of PDTC, carbon disulfide as a major product, and increased chloroform. Titanium(III) citrate addition also led to higher turnover and chloroform production. A comparison of metal additions showed that the products detected under conditions promoting transformation resembled those seen with strain KC more closely than products of hydrogenolysis catalysts, with the exception of cobalt/Ti(III) citrate.

[0148] Structure of the Cu:PDTC complex. PDTC is known to form stable complexes with iron, cobalt and nickel. Hexacoordinate (i.e., coordinatively saturated) metal ion:PDTC complexes comprised of one metal atom and two PDTC ligands have been described for Fe(II, III), Ni(II), and Co(III) using X-ray diffraction spectroscopy. In these complexes the metal atom lies in the center of octahedrally arranged ligand atoms formed by two planar PDTC molecules arranged perpendicular to each other. Using negative ion electrospray mass spectrometry (ES⁻ MS), the following molecular ions were observed: [Fe(II)(PDTC)₂]⁻², m/z 225; [Fe(III):(PDTC)₂]⁻¹, m/z 450; [Co(PDTC)₂]⁻¹, m/z 453. Structures for Co[I] and Co[II] complexes with PDTC have not been determined. Palladium is known to form a planar tetracoordinate 1:1 complex with PDTC that can also include a halide ion. Cu(II) also forms a 1:1 complex with PDTC in which copper can be coordinated by a halide ion. The ES⁻ MS analysis of CuCl:PDTC showed molecular ions: m/z 295, [⁶³Cu³⁵Cl:PDTC]⁻¹, 100%; m/z 297, [⁶⁵Cu³⁵Cl+⁶³Cu³⁷Cl:PDTC]⁻¹, 84.45% ; m/z 299, [⁶⁵Cu³⁷Cl:PDTC]⁻¹, 18.85%, (example in FIG. 5B). Elemental analysis of the Cu(II)—Br:PDTC complex also confirmed 1:1 stoichiometry. EPR spectra of the Cu—Br:PDTC complex are typical of Cu(II) complexes. A Cu(II) oxidation state assignment is also supported by elemental analysis which indicates one tetrabutylammonium cation per Cu—Br:PDTC anion.

[0149] Structures of the relevant complexes indicated that metal ions known to be active in reductive dechlorination of CCl₄ in other coordination complexes (i.e., Fe(II), Co(II), and Ni(II) in heme, corrins, and F₄₃₀, respectively) were not active when complexed with PDTC due to steric and/or electronic effects imparted by the sulfur and nitrogen atoms surrounding the metal center. Ti(III) citrate was the only reductant used that is likely to produce Co(I), which occurs as a planar tetracoordinate complex in vitamin B₁₂. An inner-sphere electron transfer process has been described for dechlorination by Fe(II) porphyrins. The data from PDTC-transition metal complexes are consistent with such a process in that only when a 1:1 complex was demonstrated (Cu(II):PDTC) or predicted (Co(I):PDTC) was dechlorination activity observed. The data showing no dechlorination activity by iron, cobalt, or nickel complexes (i.e., without reductant or cells; Table 5) suggested a model whereby only PDTC complexes having an accessible metal atom are active. The Co(II) complex having the highest stability, followed by Cu(II), best explained the data. Cu(II) could displace Fe(III) or Ni(II) and the dechlorination activity seen in the presence of iron and nickel would likely have been due to contaminating copper.

[0150] Dechlorination of CCl₄ by PDTC-transition metal complexes. To explain the observed products, a reaction pathway outlining events likely to occur after the initial encounter between CCl₄ and the metal atom has been formulated (see FIG. 4). The mechanism involves atom transfer and is reductive, with an initial one-electron transfer to CCl₄ from the Cu:PDTC complex. In this pathway CCl₄ is converted to CO₂ and HCl, and PDTC to DPA and H₂S. CS₂ is a byproduct. The critical difference between reduction by Co[I]:PDTC and the pathway of FIG. 4 would be that oxidation of Co[II] would not lead to oxidation of the neighboring sulfur atom. The oxidation of Cu:PDTC provides [Cu:PDTC]., whereas oxidation of Co(I):PDTC provides Co(II):PDTC. Evidence for oxidation of sulfur ligand atoms without net oxidation of a coordinated metal atom has been described. The oxidizing trichloromethyl radical formed by one-electron reduction of CCl₄ would be expected to have different fates under the two circumstances; with a radical in close proximity condensation of the two radicals to form a covalent bond would dominate. Without this proximal radical, reactions yielding chloroform via reduction and protonation or hydrogen abstraction are favored, explaining the higher chloroform yield with Co/Ti(III) in Table 6. The presence of reducing agents increased the likelihood of reduction of the radical and hydrogenolysis, a radical-scavenging effect. The reductants will likely reduce Cu(II) to Cu(I), which could allow some catalytic reduction of CCl₄ by the Cu:PDTC complex. An additional catalytic effect would be derived from replacement of the hydroxide in FIG. 5 with thiolate (HS⁻) as the attacking nucleophile. This would result in regeneration of the active agent, whereas hydrolysis would destroy an element of the structure required for activity by replacing sulfur with oxygen. These results are supported by the stoichiometry data of Table 6 in that the non sulfur-containing reductant, Ti(III) citrate, was less effective at increasing turnover than was sulfide.

[0151] Mass balance analysis. End product analyses were used to further assess how well the chemically defined reaction conditions represented CCl₄ transformation by strain KC. Mass balance determinations using ¹⁴CCl₄ radiotracer were used initially and showed that Cu:PDTC without added reducing agent gave a product profile consisting of approximately 65% ¹⁴CO₂, 15% non-volatile material, and 20% volatile organic-trapped products (see Table 7 below). Addition of Na₂S resulted in a substantial increase in the volatile organic-trapped product fraction and a decrease in ¹⁴CO₂ (see Table 7 below). Headspace GC/MS analysis detected carbon disulfide (CS₂) and chloroform (see Table 6 below), as well as COS as products likely to be trapped in the organic scintillant used. Those data indicated that the organic-trapped ¹⁴C was mostly ¹⁴CS₂. Using ¹³CCl₄ as a tracer confirmed that the carbon atoms of CS₂ and COS were derived from CCl₄ and not from rearrangements of PDTC. Less COS was seen when Na₂S was added. These data are consistent with predictions set forth in the pathway of FIG. 4; i.e., that CO₂ was derived from hydrolysis of thiophosgene via COS, and that the nucleophilic thiolate ion reacted with the electrophilic thiophosgene to give CS₂ at the expense of CO₂ and COS production. Comparing the data to those obtained from strain KC cultures, more CO₂ and organic-trapped products and fewer non-volatile products were seen with Cu:PDTC. These results can be explained by the presence of active nucleophiles other than HS⁻ in cultures versus the chemically-defined conditions.

[0152] Non-volatile product analysis. The non-volatile material remaining in concentrated reactions containing 2 mM Cu:PDTC and excess CCl₄ was analyzed by ES⁻ MS. Since sulfur-substitution was seen in reactions where PDTC was the only sulfur compound included, the presence of a de-sulfurylated PDTC derivative was anticipated. Reactions were performed in DMF:water to improve solubility of synthetic Cu:PDTC and facilitate ionization in electrospray MS. These reactions showed substantial bleaching of the green color of Cu:PDTC (λmax: 400 nm, 610 nm) as a result of CCl₄ addition, giving a pale blue solution (λmax: 333 nm, 710 nm) with the appearance of a brown precipitate. These results were observed whether the reactions were conducted anoxically or under an air headspace with air-equilibrated solvent. Mass spectra showed that the concentration of Cu:PDTC decreased, and that Cu:dipicolinate and an ion attributed to the partial hydrolysis product, picolinic acid-6-thiocarboxylic acid, appeared (see FIG. 5). When the reactions were performed in phosphate buffer (i.e., no DMF) with catalytic amounts of copper (1 mole %), dipicolinic acid was the major aromatic component seen by HPLC. Dipicolinic acid accounted for approximately 70% of the PDTC included in the reaction after 48 hours.

[0153] Evidence for radical intermediates. Some of the reaction products observed with Cu:PDTC/CCl₄ (CS₂, CO₂) have been predicted to arise from biotite/sulfide/CCl₄ mixtures through a radical substitution mechanism. The pathway of FIG. 4 also includes trichloromethyl radical as an intermediate. Oxygen is known to react with trichloromethyl radical at rates near diffusion limitation. The pathway proposed would predict that the presence of O₂ would divert a significant portion of the carbon flow away from sulfur-substitution products. The data of Tables 5 and 6 and FIG. 3 were obtained from anoxic conditions. When reactions were conducted under an air headspace with air-equilibrated buffer, carbon disulfide accounted for less than 0.4% of the CCl₄ transformed, whereas in a parallel experiment conducted anoxically, it accounted for 15.7% (13 μM CuCl₂, 26 μM PDTC, 4 hour incubation). The turnover of CCl₄ per PDTC in those experiments was 1.08±0.02 (mean±s.d., n=3) under air, and 1.45±0.08 anoxically. Thiols are subject to autoxidation, which can be catalyzed by transition metal ions. Solutions of PDTC (5 mM) with CuCl₂ (50 μM) showed an O₂-dependent degradation as evidenced by formation of a precipitate after several hours in air. Competition between O₂ and CCl₄ for oxidation of Cu:PDTC is therefore likely; however, it did not prevent dechlorination from occurring when exposure to CCl₄ was initiated shortly after exposure to O₂.

[0154] Additional experiments were conducted to characterize possible radical intermediates of the Cu:PDTC/CCl₄ reaction. Using EPR spectroscopy, reactions performed in phosphate buffer with catalytic amounts of CuCl₂ and excess PBN spin trap exhibited a resonance centered at g=2.008 with hyperfine coupling constants of A_(α) ^(N) 15.3 G, and A_(β) ^(H) 2.7 G. Nitroxyl radicals typically exhibit a basic triplet pattern due to nitrogen hyperfine (I=1) coupling to the radical. With additional hyperfine coupling to one hydrogen (I=½), as found in PBN trapped species, a triplet of doublets pattern is observed. To obtain definitive evidence for trapping of .CCl₃ it is convenient to introduce a further hyperfine signature through ¹³C labeling (I=½). A characteristic triplet of doublet of doublets pattern is then observed for PBN trapped .¹³CCl₃ as shown in FIG. 6. Measured hyperfine coupling constants of A₆₂ ^(13C) 8.4 G, A_(α) ^(N) 15.3 G, and A_(β) ^(H) 2.6 G were consistent with previously determined values for [¹³C trichloromethyl]-PBN spin adduct, definitively showing that trichloromethyl radical had arisen from reaction between CCl₄ and PDTC with copper. In light of the potent reducing ability of SH-containing species, we suspected that some of the PBN-trichloromethyl radical spin adduct might be reduced to an EPR-silent hydroxylamine form. Addition of 1.5 mM potassium ferricyanide as a mild oxidant led to a more than 20-fold increase in the signal shown in FIG. 6, while addition of 10 mM potassium ferricyanide caused most of the trapped signal to disappear.

[0155] Another approach to the identification of possible radical intermediates included the use of the stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) in reaction mixtures. Positive electrospray ionization MS showed the presence of 2,2,6,6-tetramethylpiperidinium cation (m/z=142) and a cation corresponding to a tetramethylpiperidine fragment (m/z=140) (see FIG. 8). Both of those species would be predicted from reactions in which a thiyl radical had condensed with the nitroxyl-containing compound (TEMPO) and decomposed through any of a variety of routes, either spontaneously or in the MS ion source.

[0156] In order to assess the viability of PDTC-dependent dechlorination as a remediation technology, it was useful to understand the underlying chemistry. The model presented can explain the product analysis and intermediate trapping data (Table 7) obtained from both P. stutzeri KC cultures and chemical reactions including PDTC and Cu[II]. This makes it possible to predict potential outcomes under various in situ conditions. One concern was that the process would not operate if iron concentrations were greater than approximately 10⁻⁵ M. The data indicate that iron should not directly inhibit dechlorination, that dechlorination instead would be limited by the amount of available copper. The amount of copper required to stimulate maximal dechlorination would be lower in the presence of metabolically active bacterial cells or reducing agents. The inhibition of dechlorination seen for ferric or ferrous iron with cultures of strain KC is therefore most likely the result of repression of PDTC synthesis, rather than a direct effect on the chemical transformation, a situation relevant to remediation since iron is likely to be more abundant than copper in most contaminated media. In addition, the use of engineered strains that are not subject to iron repression would be futile if the effect of iron was more direct. The production of so-called “exacerbating factors” by bacteria in contaminated media could confound efforts to use this transformation effectively; however, no evidence for this effect using strain KC cultures, culture supernatants, or another strain of the same species (Table 5) has been found. TABLE 5 Effects of transition metal additions, bacterial cells, and chemical reducing agents on PDTC-dependent CCl₄ transformation No 0.5 mM 0.5 mM reductant^(a) Cells^(b) Sulfide^(a) Ti[III]^(a) No PDTC  10.3 ± 16.8 28.3 ± 1.8 209^(c) 155.9 ± 4.8 added −PDTC 0 −0.4 ± 0.1  36.3 ± 7.4  1.3 ± 14.0 metal Cu PDTC 109.8 ± 7.8   29 ± 0.2 209^(c) 186.0 ± 0.3 (II) −PDTC n.d. n.d. −16.2 ± 43.8  21.0 ± 8.6 Fe PDTC  15.5 ± 14.7 29.6 ± 0.02   201 ± 9.6 118.5 ± 17.7 (III) −PDTC n.d. n.d.  −1.1 ± 11.2  −0.9 ± 14.6 Co PDTC  −2.8 ± 1.9  0.7 ± 0.2  16.2 ± 9.4  92.0 ± 11.1 (II) −PDTC n.d. n.d.  11.8 ± 2.1  −4.7 ± 3.1 Ni PDTC  −1.0 ± 4.5 22.6 ± 4.6   204 ± 4.2 149.4 ± 4.7 (II) −PDTC n.d. n.d.  3.7 ± 3.8  7.7 ± 3.3

[0157] TABLE 6 CCl₄ transformation by PDTC with or without transition metals and chemical reducing agents^(a) No reductant 0.5 mM Na₂S Ti(III) citrate CCl₄/mole^(b) % CHCl₃ % CS₂ CCl₄/mole^(b) % CHCl₃ % CS₂ CCl₄/mole^(b) % CHCl₃ % CS₂ PDTCH₂   0.2 ± 0.3 n.f. n.f. ≧4 4.5 ± 0.3 50.4 ± 2.9 3.1 ± 0.1  5.1 ± 0.8 6.3 ± 0.5 Cu:PDTC   2.2 ± 0.2 <0.1 7.5 ± 0.8 ≧4 0.9 ± 0.05 63.3 ± 5.2 ≧3.7  0.2 ± 0.1 4.5 ± 0.5 (1.85 ± 0.1)* (6.71 ± 0.1)* Fe:PDTC   0.3 ± 0.3 n.f. n.f. ≧4 5.2 ± 0.6 53.5 ± 4.6 2.4 ± 0.4  7.8 ± 0.9 8.7 ± 1.0 Co:PDTC <0.1 n.f. n.f.   0.3 ± 0.2 <0.7  3.9 ± 8.9 1.8 ± 0.2 34.2 ± 0.3 n.f. Ni:PDTC <0.1 n.f. n.f. ≧4 1.6 ± 0.3 39.6 ± 2.7 3.0 ± 0.1  2.4 ± 0.8 3.2 ± 0.9

[0158] TABLE 7 Recovery of ¹⁴C from ¹⁴CCl₄ after reaction with Cu:PDTC Non- Volatile, strippable, Total 14CO₂[₂?] organic-trapped aqueous Recovery Cu:PDTC, 68.3 ± 1.0% 20.0 ± 0.5% 8.1 ± 0.2% 96.4% 50 μM Cu:PDTC, 16.4 ± 1.4% 70.2 ± 4.2% 1.3 ± 0.2% 87.9% 50 μM 0.5 mM Na2S

[0159] Reactions were conducted in 1 ml 35 mM potassium phosphate buffer, pH 7.7, and included approximately 50 nmoles of ¹⁴CCl₄.

Example 4

[0160] In this example, the stability constants and relative binding strengths for PDTC and several of the physiologically important metals that it binds are described.

[0161] Materials. All the commercial reagents were of the highest purity available and were used without further purification. Inductively Coupled Plasma Emission (ICP) standard metal stock solutions (1 g/L per metal ion) of Cu²⁺, Fe³⁺, Co³⁺, Ni²⁺, Zn²⁺, and Mn²⁺ were obtained from Fisher Scientific (FS). These standards contained 2% nitrate to keep the metals in solution. A Cr³⁺ stock solution was made with Cr₂(SO₄)₃ suspended in 2% HNO₃. PDTC was synthesized using the method of Hildebrand et al. PDTC stock solutions were prepared by weighing out 0.100 g of PDTC and diluting it volumetrically to 10 ml with dimethylformamide (DMF). Standardized NaOH (FS SS266-1) and HCl (FS SA48-1) solutions degassed in argon were used for titrations and pH adjustments. The spectrophotometric competition studies were carried out with stock solutions of disodium ethylenediaminetetraacetate dihydrate (EDTA) (Bio-Rad 161-0728), 2,6-pyridinedicarboxylic acid (DPA) (Aldrich P6, 380-8), and K₃FeCN₆ (Sigma P-83131).

[0162] General Instruments. Absorption spectra were recorded on a Hewlett-Packard 8453 UV/Visible diode array spectrophotometer control by a HP Pentium-class computer. A Fisher Scientific Accument Basic pH meter equipped with an Accument Basic pH/ATC combination silver/silver chloride reference electrode was used for pH measurements. Mass spectra were taken with an electrospray ionization tandem mass spectrometer (Quattro II, Micromass Ltd., UK). Volume-dependent titrations employed a 665 Dosimat Metrohm volume dispenser (Brinkman Instruments Inc. Westbury N.Y.).

[0163] Potentiometric Titrations. All solutions were prepared with deionized distilled water of better than 18 M Ohm resistance, and measurements were made at 25° C. The ionic strength in the titration experiments was fixed at 0.1 M with NaClO₄ (FS SS266-1). The electrode was calibrated to read pH according to the standard method. Potentiometric titration employed the use of the automatic dispenser and the pH meter. Argon-saturated solutions were titrated with 0.1 N NaOH. The titrated sample was continually purged with argon to minimize interference of air-derived CO₂. Temperature was maintained at 25° C.

[0164] Spectrophotometric Titrations. UV-Visible absorption spectra were recorded using a 1.0-cm path length quartz cell. Spectra were analyzed on a Hewlett Packard computer running UV-Visible Chemstation software (revision 52). Concentrations of metal ions and PDTC examined ranged from 10⁻⁴ to 10⁻⁵ M.

[0165] Ligand Protonation Constants. PDTC has three protonation sites, one on the pyridine nitrogen and two on carbonyl sulfur atoms. They are denoted LH₁, LH₂, and LH₃, respectively. Precipitation of PDTC will occur depending on PDTC concentration and pH. Precipitation is favored as the concentration of PDTC increases and pH approaches 2. The protonation constants for PDTC were determined by potentiometric titration (FIG. 8). As indicated by the titration, the pK₁ is 5.48 and pK₂ is 2.58, where: $K_{n} = \frac{\left\lbrack {LH}_{n} \right\rbrack}{\left\lbrack H^{+} \right\rbrack \left\lbrack {LH}_{n - 1} \right\rbrack}$

[0166] These constants were obtained using the computer program BigBest, which gave a sigma fit of 0.055. The third protonation constant is estimated to be 1.3. FIG. 9 shows the stepwise protonation of PDTC.

[0167] Changes in absorption spectra during titration of PDTC by NaOH are presented in FIG. 10. A profound color change near the first protonation constant was observed, and is probably due to the protonation of the nitrogen atom on the pyridine ring. PDTC absorption spectra from 230 to 430 nm were strongly affected as solution pH changed from acidic to basic.

[0168] Ferric Complexes. The first analysis of the stability constant for the PDTC ferric complex was done using spectrophotometric ligand-ligand competition methods. Solutions of EDTA, dipicolinic acid (DPA), and K₃FeCN₆ were prepared for competitive studies. Results showed that EDTA and DPA could not compete with PDTC. However, it was shown that PDTC could not compete with K₃FeCN₆ for iron. The results of these experiments indicated that the complex had an overall stability constant greater than the stability constant of 10¹⁶ for DPA, yet a weaker stability constant than the overall stability constant of 10⁵² for cyanide.

[0169] A titration curve of the ferric PDTC complex yielded a reversible spectral change over the pH range 11 to 12 (FIG. 10). With no other competing ligands present, hydroxide is the competing ligand causing a spectral change from the Fe(PDTC)₂ spectrum to the spectrum of free PDTC. Plotting the spectral change at a specific wavelength, fitting a curve, taking the derivative of this curve twice, and setting it equal to zero yields the point of inflection (FIG. 12), found to occur at pH=11.43.

[0170] At this pH, 2[PDTC]=[Fe(PDTC)₂]. Iron hydroxides are known to form the following complexes: Fe(OH)²⁺, Fe(OH₂)¹⁺, Fe(OH)₃, Fe(OH)₄ ¹⁻, Fe(OH)₅ ²⁻. To a negligible extent, they also form Fe₂(OH)₂ ⁴+ and Fe₃(OH)₄ ⁵⁺. The total amount of metal present is known, and can be defined by the following relation:

T _(Fe) _(³⁺) =[Fe(pdtc)₂ ¹⁻]+[Fe(OH)₁ ²⁺]+[Fe(OH)₂ ¹⁺]+[Fe(OH)₃]+[Fe(OH)₄ ¹⁻]+[Fe(OH)₅ ²⁻]+[Fe³⁺]

[0171] Overall formation constants for iron hydroxides have been previously determined: $\begin{matrix} {\beta_{xy} = \frac{{\left\lbrack {{Fe}_{x}({OH})}_{y}^{{({3 - y})} +} \right\rbrack \left\lbrack H^{+} \right\rbrack}^{x}}{\left\lbrack {Fe}^{3 +} \right\rbrack^{x}}} & (1) \end{matrix}$

[0172] And with the substitution of the above equations: $\begin{matrix} \begin{matrix} {T_{{Fe}^{3 +}} = {\left\lbrack {{Fe}({pdtc})}_{2}^{1 -} \right\rbrack + \frac{\beta_{1}\left\lbrack {Fe}^{3 +} \right\rbrack}{\left\lbrack H^{+} \right\rbrack} + \frac{\beta_{12}\left\lbrack {Fe}^{3 +} \right\rbrack}{\left\lbrack H^{+} \right\rbrack^{2}} +}} \\ {{\frac{\beta_{13}\left\lbrack {Fe}^{3 +} \right\rbrack}{\left\lbrack H^{+} \right\rbrack^{3}} + \frac{\beta_{14}\left\lbrack {Fe}^{3 +} \right\rbrack}{\left\lbrack H^{+} \right\rbrack^{4}} + \left\lbrack {Fe}^{3 +} \right\rbrack}} \end{matrix} & (2) \end{matrix}$

[0173] Solving the above equation for the iron concentration: $\begin{matrix} {\left\lbrack {Fe}^{3 +} \right\rbrack = \frac{\left( {{- T_{{Fe}^{3 +}}} + \left\lbrack {{Fe}({pdtc})}_{2}^{1 -} \right\rbrack} \right) \times \left\lbrack H^{+} \right\rbrack^{4}}{{\beta_{11}\left\lbrack H^{+} \right\rbrack}^{3} + {\beta_{12}\left\lbrack H^{+} \right\rbrack}^{2} + {\beta_{13}\left\lbrack H^{+} \right\rbrack} + \beta_{14} + \left\lbrack H^{+} \right\rbrack^{4}}} & (3) \end{matrix}$

[0174] And knowing that iron forms the following complexes with PDTC: $\begin{matrix} {K_{11} = {{\frac{\left\lbrack ({Fepdtc})^{1 +} \right\rbrack}{\left\lbrack {Fe}^{3 +} \right\rbrack \left\lbrack {pdtc}^{2 -} \right\rbrack}\quad {and}\quad K_{12}} = \frac{\left\lbrack {{Fe}({pdtc})}_{2}^{1 -} \right\rbrack}{\left\lbrack ({Fepdtc})^{1 +} \right\rbrack \left\lbrack {pdtc}^{2 -} \right\rbrack}}} & \left( {3,4} \right) \end{matrix}$

[0175] Or in terms of overall stability constant: $\begin{matrix} {\beta_{12} = \frac{\left\lbrack {{Fe}({pdtc})}_{2}^{1 -} \right\rbrack}{{\left\lbrack {Fe}^{3 +} \right\rbrack \left\lbrack {pdtc}^{2 -} \right\rbrack}^{2}}} & (5) \end{matrix}$

[0176] With [PDTC], [Fe(PDTC)₂], and [Fe³⁺] known, β₁₂=β_(Fe+3) can now be calculated. The binding constant for the ferric PDTC complex, log β_(Fe+3), was calculated to be 33.93.

[0177] Titrations with H₂SO₄. As shown in equation 5, the determination of β₁₂ depends on concentration of [pdtc²⁻]. The concentration of PDTC is also dependent upon the first, second, and third protonation constants: $\begin{matrix} {K_{n}^{H} = \frac{\left\lbrack {H_{n}{pdtc}} \right\rbrack}{\left\lbrack {H_{n - 1}{pdtc}} \right\rbrack \left\lbrack H^{+} \right\rbrack}} & (6) \end{matrix}$

[0178] Therefore, the hydrogen ion competes with any metal PDTC complex. With successive additions of hydrogen ion, it becomes possible to find the point at which H⁺ outcompetes the metal ion. Since the protonation constants were previously determined, it becomes possible to estimate the stability constant based on the hydrogen concentration. The free iron concentration can be defined as: $\begin{matrix} {\left\lbrack {Fe}^{3 +} \right\rbrack = {T_{{Fe}^{3 +}} \times \frac{\left\lbrack {{Fe}\lbrack{pdtc})}_{2}^{1 -} \right\rbrack}{2}}} & (7) \end{matrix}$

[0179] A mass balance on the total ligand present yields:

T _(L)=2×[Fe(pdtc)₂ ¹⁻]+[pdtc²⁻]+[Hpdtc¹⁻]+[H₂pdtc]+[H₃pdtc¹⁺]  (8)

[0180] Using equations 8 and 6 to solve for [PDTC²⁻]: $\begin{matrix} {\left\lbrack {pdtc}^{2 -} \right\rbrack = \frac{{- \left( {{- T_{L}} + {2 \times \left\lbrack {Fepdtc}_{2}^{2 -} \right\rbrack}} \right)} \times K_{1}^{H} \times K_{2}^{H} \times K_{3}^{H}}{\left( {{K_{1}^{H} \times K_{2}^{H} \times K_{3}^{H}} + {\left\lbrack H^{+} \right\rbrack \times K_{2}^{H} \times K_{3}^{H}} + \left\lbrack H^{+} \right\rbrack^{2} + \left\lbrack H^{+} \right\rbrack^{3}} \right.}} & (9) \end{matrix}$

[0181] The concentration of [Fe(PDTC)₂] was determined spectrophotometrically using a calibration curve at a wavelength of 605 nm. Now all three components of equation 5 are known from equations 7 and 9 and the [Fe(PDTC)₂] calibration curve. The log β₁₂ was found to be 32.49, but this value has several limitations for use. In order to approach a point where hydrogen ion could compete with iron, a concentration of 7-8 molar H₂SO₄ had to be reached. At an ionic strength this high, and because PDTC is believed to undergo acid hydrolysis at this pH, the number has little value in the determination of a stability constant. However, the number generated gives a fairly accurate estimate of the real stability constant at this extremely high ionic strength, and can be considered valuable for comparing the relative strengths of PDTC with other metal compounds. The same conditions were applied to cobalt, which gave a log β of 32.2.

[0182] Metal-Metal Competition. An array of metal solutions was set up for competition studies of metal versus metal. The experiment was set up so that PDTC could completely complex with either metal species present. Since Fe, Co, Ni, Mn and Cr form a 1:2 M(PDTC)₂ complex, the 0.25 mM solutions of two metals forming 0.5 mM were mixed with PDTC to reach a final concentration of 0.5 mM. The PDTC concentration in each bimetal solution was half that required to completely complex both metals. Table 8 shows how the experimental array was set up and also shows the dominant species for each metal in the metal competition experiment. The manganese and chromium spectra were too similar to conclude which species dominated. An equal sign indicates that both complexes for M(PDTC)₂ were present in similar proportion.

[0183] Metals with similar affinities for PDTC can be evaluated numerically for relative strengths. For metals with a 2:1 complex formation, the following relation can be used: $\begin{matrix} {\beta_{M2} = {\beta_{M1}\frac{\left\lbrack M_{1}^{z_{1}} \right\rbrack \left\lbrack {M_{2}({pdtc})}_{2}^{z_{2} - 4} \right\rbrack}{\left\lbrack M_{2}^{z_{2}} \right\rbrack \left\lbrack {M_{1}({pdtc})}_{2}^{z_{1} - 4} \right\rbrack}}} & (10) \end{matrix}$

[0184] The above equation was used to find the stability constant for the [Co(PDTC)₂]¹⁻ complex. Since the [Fe(PDTC)₂]¹⁻ complex has a distinctive absorbance peak at 605 nm, the concentration this species was measured with a calibration curve (FIG. 12). A mass balance would then yield the following relation:

T _(M) _(^(z)) =[M(pdtc)₂ ^(z−4) ]+[M ^(z)]  (11)

[0185] Using equation (10) to find each species concentration and then solving for β_(M2) in equation (11) yields the overall stability constant. It was found that log β_(Co) _(³⁺) was 33.45 and log β_(Ni) _(³⁺) was 33.56.

[0186] Potentiometric Titration. Results of the titration of PDTC show that pK₁ is 5.48 and that the pK₂ is 2.58. The program BigBest minimizes the standard deviations of fit over the entire titration curve by variation of the protonation constants. The error associated with the fit is relatively low, reported as SIGFIT=0.055. Error analysis of the third protonation constants was computed as 1.3. The determination of the third protonation constant for PDTC is an estimate at best, since titration methods are only valid at a pH range from 2 to 12 and the computation was out of the range of experimental data.

[0187] Spectrophotometric Titration. The challenge of measuring the overall equilibrium for PDTC was difficult, due to the nature of the proton dissociation constants and the high affinity of PDTC to the metals of interest. Competition studies for metabolic chelators often use EDTA as the competitor, even if EDTA has a lesser stability constant. This method relies on the fact that the complex has protonation sites with high affinities to the proton, and which also bind to the metal. As the pH is lowered, the apparent strength of the complex is weakened to a greater extent than is EDTA, so that at a certain pH, EDTA can outcompete the complex. If the pKa values of the ligand are low, and the stability constant is greater than that of EDTA, the ligand outcompetes EDTA at any pH. Thus, hydroxide was chosen as the competing ligand for measuring the stability constant.

[0188] Limitations on the accuracy of the determined stability constant included the fact that PDTC slowly undergoes base-catalyzed hydrolysis in a solution with basic pH. For this reason the time allowed for equilibrium to be reached was less then 10 minutes. It was noted, however, that very little change occurred over this time interval, indicating that the amount of time for equilibrium was sufficient.

[0189] The method used was verified by measuring the stability constant for EDTA under the same method and conditions. Hydroxide was found to compete with EDTA over the pH range of 6.5 to 9. Under the same titration conditions as in the iron(PDTC)₂ experiment, [EDTA] was found to equal [FeEDTA] at a pH of 7.63. The log K_(eff) for EDTA at this pH was calculated to be 14.9. The experimental log K_(eff) of EDTA was found to be 15.1. The calculated error of the log K_(eff FeEDTA) was then found to be 1.5%.

[0190] The results of the titration with copious amount of H₂SO₄ resulted in a log β_(Fe) _(³⁺) of 32.49 versus the log β_(Fe) _(³⁺) of 33.93. This 4% difference in log β_(Fe) _(³⁺) values is likely due to the different ionic strengths of the experiments and any possible error in the estimation of the third protonation constant.

[0191] Metal-Metal Competition Studies. The data from Table 8 can produce the following relative strengths of each metal (from strongest to weakest): Cu²⁺, Fe³⁺, Co³⁺>Ni²⁺>Zn²⁺>Mn²⁺, Cr³⁺.

[0192] Comparison with Other Iron Chelators. Reported stability constants of metabolically produced chelators can be very large. For example, enterobactin has an estimated log K_(ML) of 52. For the reasons mentioned previously, the chelating power of enterobactin is weakened as the pH is lowered. The low pKa values of PDTC, however, give it the ability to outcompete enterobactin below a pH of 6.6. A comparison of various chelators with PDTC is shown in Table 9. TABLE 8 Results of cross competition metal/metal study for pdtc. [Fe³⁺], [Co³⁺], [Ni²⁺], [Cr³⁺] = 0.25 mM; [Cu²⁺], [Zn²⁺], [Mn²⁺] = 0.5 mM; [pdtc] = 0.5 mM; in 2% HNO₃ ; T = 25° C. For ?, spectra too similar to determine which metal complex was formed; for =, both complexes were present in similar proportions. Co³⁺ Ni²⁺ Cu²⁺ Zn²⁺ Mn²⁺ Cr³⁺ Fe³⁺ = Fe³⁺ = Fe³⁺ Fe³⁺ Fe³⁺ Co³⁺ = = Co³⁺ Co³⁺ Co³⁺ Ni²⁺ Cu²⁺ Zn²⁺ = Ni²⁺ Cu²⁺ Cu²⁺ Cu²⁺ Cu²⁺ Zn²⁺ Zn²⁺ Zn²⁺ Mn²⁺ ?

[0193] TABLE 9 Log β values for various iron chelates Ligand log β Mecam 35.6 pdtc 34.0 Ferrioxamine E 32.9 Ferrochrome A 32.0 Ferrichrome 29.1 EDTA 25.0 DPTA 27.6 Ferrichrysin 26.5 EDTA 25.0

[0194] In summary, the spectrophotometric and potentiometric titration studies on ferric PDTC show its strong affinity for Fe³⁺. Moreover, PDTC has been found to have comparable affinities for various other metals. The log stability constants for the iron III, cobalt III, and nickel II PDTC complexes have been found to be 33.93, 33.28, and 33.36, respectively. The first and second protonation constants for PDTC were found to be 5.48 and 2.58. The third pKa was estimated to be 1.3. These protonation constants and high affinity constants show that over a physiological pH range, ferric PDTC has one of the highest effective overall stability constants for metal binding among known bacterial chelators.

[0195] While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 35 <210> SEQ ID NO 1 <211> LENGTH: 2061 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2061) <223> OTHER INFORMATION: ORF K <400> SEQUENCE: 1 ctg ggc cgg ggc agt cga ctc ccg agc gca gct tac ggg agt acg cat 48 Leu Gly Arg Gly Ser Arg Leu Pro Ser Ala Ala Tyr Gly Ser Thr His 1 5 10 15 gat atg cgc ggt caa ccg atg atg atg gct aca gct ttg atc tgt gcc 96 Asp Met Arg Gly Gln Pro Met Met Met Ala Thr Ala Leu Ile Cys Ala 20 25 30 ttt gta cca ggg cca cag ttg gcg ttt gct gcg cca ggc tcc gcg gct 144 Phe Val Pro Gly Pro Gln Leu Ala Phe Ala Ala Pro Gly Ser Ala Ala 35 40 45 tcg cct gac tcc acg acg cta ccg gaa atc acc gtc aca gcc gag aaa 192 Ser Pro Asp Ser Thr Thr Leu Pro Glu Ile Thr Val Thr Ala Glu Lys 50 55 60 atc gag cgg ccg ctg gaa agg gtg ccc gcc agc gtg gcg gtg atc gat 240 Ile Glu Arg Pro Leu Glu Arg Val Pro Ala Ser Val Ala Val Ile Asp 65 70 75 80 ggc tgg gac gcc gag cag tca ggc atc act agc ctc aaa caa ctg gaa 288 Gly Trp Asp Ala Glu Gln Ser Gly Ile Thr Ser Leu Lys Gln Leu Glu 85 90 95 gga cgc att cct ggt ctg tca ttc cag ccg ttc ggg caa gca ggt atg 336 Gly Arg Ile Pro Gly Leu Ser Phe Gln Pro Phe Gly Gln Ala Gly Met 100 105 110 aat tca ccc gtc atg cgg ggg ctg acg gcc aac ttc aac agc ttc tcc 384 Asn Ser Pro Val Met Arg Gly Leu Thr Ala Asn Phe Asn Ser Phe Ser 115 120 125 agt tca acg ttg ttg ctg gtc gat ggc gtt ccc acg ctg aca gcc cag 432 Ser Ser Thr Leu Leu Leu Val Asp Gly Val Pro Thr Leu Thr Ala Gln 130 135 140 gga ttc gag agt ggc atg ctg gat ctc gat cgc atc gag gtc att cgc 480 Gly Phe Glu Ser Gly Met Leu Asp Leu Asp Arg Ile Glu Val Ile Arg 145 150 155 160 ggc ccg caa tct acg ctg tat ggc cgt aat gcc gag gcc ggt gtg att 528 Gly Pro Gln Ser Thr Leu Tyr Gly Arg Asn Ala Glu Ala Gly Val Ile 165 170 175 gcc atc cac agc ctg ccg atg gac gcg acc ccg aga gcc agc gtg tct 576 Ala Ile His Ser Leu Pro Met Asp Ala Thr Pro Arg Ala Ser Val Ser 180 185 190 gcc gaa gcg ggc agc cgg aac aag cgt gtc atg cgg ttt gcg ctc agc 624 Ala Glu Ala Gly Ser Arg Asn Lys Arg Val Met Arg Phe Ala Leu Ser 195 200 205 cag cct ttg gtg gaa gag cgg ttg tac ggc agc gta tcg ggc aac tgg 672 Gln Pro Leu Val Glu Glu Arg Leu Tyr Gly Ser Val Ser Gly Asn Trp 210 215 220 tcg agc cag gac ggc ttc atc gac aac acc cac acg ggg cac aag gcg 720 Ser Ser Gln Asp Gly Phe Ile Asp Asn Thr His Thr Gly His Lys Ala 225 230 235 240 gac gat cgt gag cag aar aac ctg aac ctg ggg ctg cgc tgg gcc ccg 768 Asp Asp Arg Glu Gln Lys Asn Leu Asn Leu Gly Leu Arg Trp Ala Pro 245 250 255 ggg gcc gca acg gat gtg gtc atg cgc tat gcg cat cag gag tac gac 816 Gly Ala Ala Thr Asp Val Val Met Arg Tyr Ala His Gln Glu Tyr Asp 260 265 270 gat ggc gcc tcc ctg tgg ggc tcg ccc ggc gcg cca agg aag cga gtc 864 Asp Gly Ala Ser Leu Trp Gly Ser Pro Gly Ala Pro Arg Lys Arg Val 275 280 285 gcg tcc gga acg ccg agc tgg aac cgt tct gag ggc cag acc ttg tcc 912 Ala Ser Gly Thr Pro Ser Trp Asn Arg Ser Glu Gly Gln Thr Leu Ser 290 295 300 ttc aat gtc cag cat gaa ttt gcc tcc ggc ctg cgg ttg cat tcg gta 960 Phe Asn Val Gln His Glu Phe Ala Ser Gly Leu Arg Leu His Ser Val 305 310 315 320 acg gcc tgg aac gag ttc aag gac agg att cag cag gac act gac ttc 1008 Thr Ala Trp Asn Glu Phe Lys Asp Arg Ile Gln Gln Asp Thr Asp Phe 325 330 335 atg cca gcc gat gtt ctg cac gtc ggg cgc gac cat cac ctg cgc aca 1056 Met Pro Ala Asp Val Leu His Val Gly Arg Asp His His Leu Arg Thr 340 345 350 ctc tcc cag gag ttc cgt gtg gag gga cag ctc ggg gag gcc agt tgg 1104 Leu Ser Gln Glu Phe Arg Val Glu Gly Gln Leu Gly Glu Ala Ser Trp 355 360 365 ctg gct ggt gtc tac gcg gat cgc agc gac aac gat ctg cac agt acc 1152 Leu Ala Gly Val Tyr Ala Asp Arg Ser Asp Asn Asp Leu His Ser Thr 370 375 380 agc aag acc atg atg ggg ctg tcg gac att cgc gcg gat cag cag agc 1200 Ser Lys Thr Met Met Gly Leu Ser Asp Ile Arg Ala Asp Gln Gln Ser 385 390 395 400 gat acc gct gca ctg ttc acc cac tgg aac gtc ccc ctg tcg gcc gac 1248 Asp Thr Ala Ala Leu Phe Thr His Trp Asn Val Pro Leu Ser Ala Asp 405 410 415 tgg tcc ata gac gcc gga gcg cgc gtc gag cgc aac gag gtg cag cta 1296 Trp Ser Ile Asp Ala Gly Ala Arg Val Glu Arg Asn Glu Val Gln Leu 420 425 430 cgt ccg caa ggg gct acg agc cat gaa aaa ggc tgg aca cac gtt tca 1344 Arg Pro Gln Gly Ala Thr Ser His Glu Lys Gly Trp Thr His Val Ser 435 440 445 ccc agg ctc gcg ctg caa cac cag ata acc gcc aat cac caa tgg tat 1392 Pro Arg Leu Ala Leu Gln His Gln Ile Thr Ala Asn His Gln Trp Tyr 450 455 460 gtg agt gcc agt cgt ggc gtg cgc act ggc ggc ttc aat gtg ctg gcg 1440 Val Ser Ala Ser Arg Gly Val Arg Thr Gly Gly Phe Asn Val Leu Ala 465 470 475 480 ccg acg ctg ggt tat ctg cct tac gac acg gag aag aac tgg tcg tat 1488 Pro Thr Leu Gly Tyr Leu Pro Tyr Asp Thr Glu Lys Asn Trp Ser Tyr 485 490 495 gaa acc ggt ctc aag ggc tgg ctt ctt gac aag cgc att cgc tat tcg 1536 Glu Thr Gly Leu Lys Gly Trp Leu Leu Asp Lys Arg Ile Arg Tyr Ser 500 505 510 ctg gcc gcc tac ctc atg gac atc gat gac atg cag gtc atg cag atg 1584 Leu Ala Ala Tyr Leu Met Asp Ile Asp Asp Met Gln Val Met Gln Met 515 520 525 ccc acc gtc ggc gtg atg tac atc acc agc gct gcc acg gcg aca tcc 1632 Pro Thr Val Gly Val Met Tyr Ile Thr Ser Ala Ala Thr Ala Thr Ser 530 535 540 aaa ggt ctc gag ctg gat gtg gac tat ctc ctc ggt ggc ggc tgg cag 1680 Lys Gly Leu Glu Leu Asp Val Asp Tyr Leu Leu Gly Gly Gly Trp Gln 545 550 555 560 ctc aag ggc ggg ctg gcc tgg aac cac acg cgc ttc gat cac ttt cgc 1728 Leu Lys Gly Gly Leu Ala Trp Asn His Thr Arg Phe Asp His Phe Arg 565 570 575 gat ggc gag gcg gac tat gac ggc aac cag aac ccg ttc gcg ccg gat 1776 Asp Gly Glu Ala Asp Tyr Asp Gly Asn Gln Asn Pro Phe Ala Pro Asp 580 585 590 ctc acc ggc cac ctc ggc atc cgc tac gac gcg ccc gaa ggc tgg tat 1824 Leu Thr Gly His Leu Gly Ile Arg Tyr Asp Ala Pro Glu Gly Trp Tyr 595 600 605 gca caa gcc agc gtg acc ggc agc agc aag gtc tac ctc gat gcg gcc 1872 Ala Gln Ala Ser Val Thr Gly Ser Ser Lys Val Tyr Leu Asp Ala Ala 610 615 620 aac ggg tat gaa cgc aac ggc tac ggc ctg gtg aac ctg gta gct ggt 1920 Asn Gly Tyr Glu Arg Asn Gly Tyr Gly Leu Val Asn Leu Val Ala Gly 625 630 635 640 tac caa cgc ggc aac tgg gaa atc gcg gcc tac gcc gac aac gcg acc 1968 Tyr Gln Arg Gly Asn Trp Glu Ile Ala Ala Tyr Ala Asp Asn Ala Thr 645 650 655 gat cag cgc tac gac gcg gtg ggc tac cag aac gga ttc gtc acc gtc 2016 Asp Gln Arg Tyr Asp Ala Val Gly Tyr Gln Asn Gly Phe Val Thr Val 660 665 670 tac agc ccg ccg cga gaa gcg ggc ctg cgt ctg aca tgg cgc ctg 2061 Tyr Ser Pro Pro Arg Glu Ala Gly Leu Arg Leu Thr Trp Arg Leu 675 680 685 <210> SEQ ID NO 2 <211> LENGTH: 687 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 2 Leu Gly Arg Gly Ser Arg Leu Pro Ser Ala Ala Tyr Gly Ser Thr His 1 5 10 15 Asp Met Arg Gly Gln Pro Met Met Met Ala Thr Ala Leu Ile Cys Ala 20 25 30 Phe Val Pro Gly Pro Gln Leu Ala Phe Ala Ala Pro Gly Ser Ala Ala 35 40 45 Ser Pro Asp Ser Thr Thr Leu Pro Glu Ile Thr Val Thr Ala Glu Lys 50 55 60 Ile Glu Arg Pro Leu Glu Arg Val Pro Ala Ser Val Ala Val Ile Asp 65 70 75 80 Gly Trp Asp Ala Glu Gln Ser Gly Ile Thr Ser Leu Lys Gln Leu Glu 85 90 95 Gly Arg Ile Pro Gly Leu Ser Phe Gln Pro Phe Gly Gln Ala Gly Met 100 105 110 Asn Ser Pro Val Met Arg Gly Leu Thr Ala Asn Phe Asn Ser Phe Ser 115 120 125 Ser Ser Thr Leu Leu Leu Val Asp Gly Val Pro Thr Leu Thr Ala Gln 130 135 140 Gly Phe Glu Ser Gly Met Leu Asp Leu Asp Arg Ile Glu Val Ile Arg 145 150 155 160 Gly Pro Gln Ser Thr Leu Tyr Gly Arg Asn Ala Glu Ala Gly Val Ile 165 170 175 Ala Ile His Ser Leu Pro Met Asp Ala Thr Pro Arg Ala Ser Val Ser 180 185 190 Ala Glu Ala Gly Ser Arg Asn Lys Arg Val Met Arg Phe Ala Leu Ser 195 200 205 Gln Pro Leu Val Glu Glu Arg Leu Tyr Gly Ser Val Ser Gly Asn Trp 210 215 220 Ser Ser Gln Asp Gly Phe Ile Asp Asn Thr His Thr Gly His Lys Ala 225 230 235 240 Asp Asp Arg Glu Gln Lys Asn Leu Asn Leu Gly Leu Arg Trp Ala Pro 245 250 255 Gly Ala Ala Thr Asp Val Val Met Arg Tyr Ala His Gln Glu Tyr Asp 260 265 270 Asp Gly Ala Ser Leu Trp Gly Ser Pro Gly Ala Pro Arg Lys Arg Val 275 280 285 Ala Ser Gly Thr Pro Ser Trp Asn Arg Ser Glu Gly Gln Thr Leu Ser 290 295 300 Phe Asn Val Gln His Glu Phe Ala Ser Gly Leu Arg Leu His Ser Val 305 310 315 320 Thr Ala Trp Asn Glu Phe Lys Asp Arg Ile Gln Gln Asp Thr Asp Phe 325 330 335 Met Pro Ala Asp Val Leu His Val Gly Arg Asp His His Leu Arg Thr 340 345 350 Leu Ser Gln Glu Phe Arg Val Glu Gly Gln Leu Gly Glu Ala Ser Trp 355 360 365 Leu Ala Gly Val Tyr Ala Asp Arg Ser Asp Asn Asp Leu His Ser Thr 370 375 380 Ser Lys Thr Met Met Gly Leu Ser Asp Ile Arg Ala Asp Gln Gln Ser 385 390 395 400 Asp Thr Ala Ala Leu Phe Thr His Trp Asn Val Pro Leu Ser Ala Asp 405 410 415 Trp Ser Ile Asp Ala Gly Ala Arg Val Glu Arg Asn Glu Val Gln Leu 420 425 430 Arg Pro Gln Gly Ala Thr Ser His Glu Lys Gly Trp Thr His Val Ser 435 440 445 Pro Arg Leu Ala Leu Gln His Gln Ile Thr Ala Asn His Gln Trp Tyr 450 455 460 Val Ser Ala Ser Arg Gly Val Arg Thr Gly Gly Phe Asn Val Leu Ala 465 470 475 480 Pro Thr Leu Gly Tyr Leu Pro Tyr Asp Thr Glu Lys Asn Trp Ser Tyr 485 490 495 Glu Thr Gly Leu Lys Gly Trp Leu Leu Asp Lys Arg Ile Arg Tyr Ser 500 505 510 Leu Ala Ala Tyr Leu Met Asp Ile Asp Asp Met Gln Val Met Gln Met 515 520 525 Pro Thr Val Gly Val Met Tyr Ile Thr Ser Ala Ala Thr Ala Thr Ser 530 535 540 Lys Gly Leu Glu Leu Asp Val Asp Tyr Leu Leu Gly Gly Gly Trp Gln 545 550 555 560 Leu Lys Gly Gly Leu Ala Trp Asn His Thr Arg Phe Asp His Phe Arg 565 570 575 Asp Gly Glu Ala Asp Tyr Asp Gly Asn Gln Asn Pro Phe Ala Pro Asp 580 585 590 Leu Thr Gly His Leu Gly Ile Arg Tyr Asp Ala Pro Glu Gly Trp Tyr 595 600 605 Ala Gln Ala Ser Val Thr Gly Ser Ser Lys Val Tyr Leu Asp Ala Ala 610 615 620 Asn Gly Tyr Glu Arg Asn Gly Tyr Gly Leu Val Asn Leu Val Ala Gly 625 630 635 640 Tyr Gln Arg Gly Asn Trp Glu Ile Ala Ala Tyr Ala Asp Asn Ala Thr 645 650 655 Asp Gln Arg Tyr Asp Ala Val Gly Tyr Gln Asn Gly Phe Val Thr Val 660 665 670 Tyr Ser Pro Pro Arg Glu Ala Gly Leu Arg Leu Thr Trp Arg Leu 675 680 685 <210> SEQ ID NO 3 <211> LENGTH: 1176 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1176) <223> OTHER INFORMATION: ORF N <400> SEQUENCE: 3 atg aac gtt gaa acc cat cgt cct gcc tcg ctg gct gga ctg tcc gcg 48 Met Asn Val Glu Thr His Arg Pro Ala Ser Leu Ala Gly Leu Ser Ala 1 5 10 15 ctt ctg ttg ata gcg atg ggc atg ccg atg atg atc ttc tat gct atc 96 Leu Leu Leu Ile Ala Met Gly Met Pro Met Met Ile Phe Tyr Ala Ile 20 25 30 ggc atc ctg ggc ccg cac ctg gtt gcc gac ttg ggg att tcc cgt cag 144 Gly Ile Leu Gly Pro His Leu Val Ala Asp Leu Gly Ile Ser Arg Gln 35 40 45 caa ctg ggc tgg ctg acc gcc agc acc ttc gga ctc gcc gcc ctg ctg 192 Gln Leu Gly Trp Leu Thr Ala Ser Thr Phe Gly Leu Ala Ala Leu Leu 50 55 60 tca ccc tgg gca ggc gca ctg gtc caa cgc atg ggc act cgt gcg ggg 240 Ser Pro Trp Ala Gly Ala Leu Val Gln Arg Met Gly Thr Arg Ala Gly 65 70 75 80 ctg ata tgc atg ttc ctg ctg gtg ggg ttg tcc ttt tcg cta atg gcg 288 Leu Ile Cys Met Phe Leu Leu Val Gly Leu Ser Phe Ser Leu Met Ala 85 90 95 gtc ctg cct ggc ttc ggt gga ttg gtc acg gca tta ctg ctt tgc ggg 336 Val Leu Pro Gly Phe Gly Gly Leu Val Thr Ala Leu Leu Leu Cys Gly 100 105 110 acg gcc cag tca ttg gca aac ccg gcg acc aat cag gcc atc gcg cat 384 Thr Ala Gln Ser Leu Ala Asn Pro Ala Thr Asn Gln Ala Ile Ala His 115 120 125 agc gta ccc gtt gcg cgg aaa gcg ggt gtc gtc ggt ctg aag cag tcg 432 Ser Val Pro Val Ala Arg Lys Ala Gly Val Val Gly Leu Lys Gln Ser 130 135 140 ggt gtg cag gcg tcc gcc ttg ttg gcg ggc gtg gcg ctt cca ccg ctg 480 Gly Val Gln Ala Ser Ala Leu Leu Ala Gly Val Ala Leu Pro Pro Leu 145 150 155 160 gtg ctg atg tgg gga tgg cgt ggc gcg ttg gca gcc tgg gtg ccc gtg 528 Val Leu Met Trp Gly Trp Arg Gly Ala Leu Ala Ala Trp Val Pro Val 165 170 175 gca ttg gtc atg gcc gca ttg gtg acc tat tgg gta cct gca aaa tcg 576 Ala Leu Val Met Ala Ala Leu Val Thr Tyr Trp Val Pro Ala Lys Ser 180 185 190 gtg tca gcg cca agc ctg cca ttg cgc gtg cgt gga ccc aat gtg tgg 624 Val Ser Ala Pro Ser Leu Pro Leu Arg Val Arg Gly Pro Asn Val Trp 195 200 205 ctg tcg ata ttg atg gcc att cag ctt tgc gca ggt ctt gcg ctg tcc 672 Leu Ser Ile Leu Met Ala Ile Gln Leu Cys Ala Gly Leu Ala Leu Ser 210 215 220 tcg ttc atg acc ttc ctc ggc gtc tat gcc gcc cag atc ggc gta tcg 720 Ser Phe Met Thr Phe Leu Gly Val Tyr Ala Ala Gln Ile Gly Val Ser 225 230 235 240 gtc agc acc atc gga gcc atg gtc agt tgc ttt ggc gcc atg ggg atc 768 Val Ser Thr Ile Gly Ala Met Val Ser Cys Phe Gly Ala Met Gly Ile 245 250 255 ctg tcc cgc gtg ctg ctg aca ccg atc gct gac aag ctg aag gac gaa 816 Leu Ser Arg Val Leu Leu Thr Pro Ile Ala Asp Lys Leu Lys Asp Glu 260 265 270 acc att ttg ctc ggc gtg ctg ttc ata ctt gca ggc ctg gcg ctg gcg 864 Thr Ile Leu Leu Gly Val Leu Phe Ile Leu Ala Gly Leu Ala Leu Ala 275 280 285 gtc atg cgc gag gcc aac act cag cag cac tgg cca ctc tgg ctt ggc 912 Val Met Arg Glu Ala Asn Thr Gln Gln His Trp Pro Leu Trp Leu Gly 290 295 300 gtg acg gga atg ggc ctg acc gtg gcc gcc agc aat gcc atc gcc atg 960 Val Thr Gly Met Gly Leu Thr Val Ala Ala Ser Asn Ala Ile Ala Met 305 310 315 320 agc atg ctg ttg cgc gat ggg cga ttt ggc ggc gca gcc act tcg gcg 1008 Ser Met Leu Leu Arg Asp Gly Arg Phe Gly Gly Ala Ala Thr Ser Ala 325 330 335 ggg atg cta tcg gtt gga ttc ttc ggc ggc ttc gcc gtt ggc ccg cct 1056 Gly Met Leu Ser Val Gly Phe Phe Gly Gly Phe Ala Val Gly Pro Pro 340 345 350 gca ttc ggg tgg ttc ttg gcc cac tca gag ggc ttc gcg gcc gcc tgg 1104 Ala Phe Gly Trp Phe Leu Ala His Ser Glu Gly Phe Ala Ala Ala Trp 355 360 365 ctt agc ctg atc ggt atc ttg gtc gca ggc ggt ctt ctg tgc ctg ctg 1152 Leu Ser Leu Ile Gly Ile Leu Val Ala Gly Gly Leu Leu Cys Leu Leu 370 375 380 ttg ctc tac atg cgc cat cgg gaa 1176 Leu Leu Tyr Met Arg His Arg Glu 385 390 <210> SEQ ID NO 4 <211> LENGTH: 392 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 4 Met Asn Val Glu Thr His Arg Pro Ala Ser Leu Ala Gly Leu Ser Ala 1 5 10 15 Leu Leu Leu Ile Ala Met Gly Met Pro Met Met Ile Phe Tyr Ala Ile 20 25 30 Gly Ile Leu Gly Pro His Leu Val Ala Asp Leu Gly Ile Ser Arg Gln 35 40 45 Gln Leu Gly Trp Leu Thr Ala Ser Thr Phe Gly Leu Ala Ala Leu Leu 50 55 60 Ser Pro Trp Ala Gly Ala Leu Val Gln Arg Met Gly Thr Arg Ala Gly 65 70 75 80 Leu Ile Cys Met Phe Leu Leu Val Gly Leu Ser Phe Ser Leu Met Ala 85 90 95 Val Leu Pro Gly Phe Gly Gly Leu Val Thr Ala Leu Leu Leu Cys Gly 100 105 110 Thr Ala Gln Ser Leu Ala Asn Pro Ala Thr Asn Gln Ala Ile Ala His 115 120 125 Ser Val Pro Val Ala Arg Lys Ala Gly Val Val Gly Leu Lys Gln Ser 130 135 140 Gly Val Gln Ala Ser Ala Leu Leu Ala Gly Val Ala Leu Pro Pro Leu 145 150 155 160 Val Leu Met Trp Gly Trp Arg Gly Ala Leu Ala Ala Trp Val Pro Val 165 170 175 Ala Leu Val Met Ala Ala Leu Val Thr Tyr Trp Val Pro Ala Lys Ser 180 185 190 Val Ser Ala Pro Ser Leu Pro Leu Arg Val Arg Gly Pro Asn Val Trp 195 200 205 Leu Ser Ile Leu Met Ala Ile Gln Leu Cys Ala Gly Leu Ala Leu Ser 210 215 220 Ser Phe Met Thr Phe Leu Gly Val Tyr Ala Ala Gln Ile Gly Val Ser 225 230 235 240 Val Ser Thr Ile Gly Ala Met Val Ser Cys Phe Gly Ala Met Gly Ile 245 250 255 Leu Ser Arg Val Leu Leu Thr Pro Ile Ala Asp Lys Leu Lys Asp Glu 260 265 270 Thr Ile Leu Leu Gly Val Leu Phe Ile Leu Ala Gly Leu Ala Leu Ala 275 280 285 Val Met Arg Glu Ala Asn Thr Gln Gln His Trp Pro Leu Trp Leu Gly 290 295 300 Val Thr Gly Met Gly Leu Thr Val Ala Ala Ser Asn Ala Ile Ala Met 305 310 315 320 Ser Met Leu Leu Arg Asp Gly Arg Phe Gly Gly Ala Ala Thr Ser Ala 325 330 335 Gly Met Leu Ser Val Gly Phe Phe Gly Gly Phe Ala Val Gly Pro Pro 340 345 350 Ala Phe Gly Trp Phe Leu Ala His Ser Glu Gly Phe Ala Ala Ala Trp 355 360 365 Leu Ser Leu Ile Gly Ile Leu Val Ala Gly Gly Leu Leu Cys Leu Leu 370 375 380 Leu Leu Tyr Met Arg His Arg Glu 385 390 <210> SEQ ID NO 5 <211> LENGTH: 1053 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1053) <223> OTHER INFORMATION: ORF P <400> SEQUENCE: 5 gtg ggt gac gta gcg ctt atg agg tcc aag atg aaa ttt caa cac gcc 48 Val Gly Asp Val Ala Leu Met Arg Ser Lys Met Lys Phe Gln His Ala 1 5 10 15 cat cca ttg caa tcc tgc tgg gat ctc gcg ctg gcg tct gtg cag gct 96 His Pro Leu Gln Ser Cys Trp Asp Leu Ala Leu Ala Ser Val Gln Ala 20 25 30 gat gcg ttg gcg act gcg ctg gat agc ggc ctt ttc gat gtg ctg acc 144 Asp Ala Leu Ala Thr Ala Leu Asp Ser Gly Leu Phe Asp Val Leu Thr 35 40 45 gaa cag gca ggg gcc ggg aaa att gca gag cag ctc gaa ctt gat ccg 192 Glu Gln Ala Gly Ala Gly Lys Ile Ala Glu Gln Leu Glu Leu Asp Pro 50 55 60 aac agg ctc gag ccc gtt ctt gag ttg tta tgg agc atg gat ctg ttg 240 Asn Arg Leu Glu Pro Val Leu Glu Leu Leu Trp Ser Met Asp Leu Leu 65 70 75 80 gag cgg ttt cag ctc cat gac agc act ggg tcg acg cca cac tat cgt 288 Glu Arg Phe Gln Leu His Asp Ser Thr Gly Ser Thr Pro His Tyr Arg 85 90 95 tcc agc gac aaa gca aag cgt ttt ttc gcc aag cag tcg tcc gat tac 336 Ser Ser Asp Lys Ala Lys Arg Phe Phe Ala Lys Gln Ser Ser Asp Tyr 100 105 110 tgc ggt gat gct tgg gcc ttt cgc ctt cgg tcg cta cgc gac ttt ggc 384 Cys Gly Asp Ala Trp Ala Phe Arg Leu Arg Ser Leu Arg Asp Phe Gly 115 120 125 acg cga ttg ccg gaa tat ctg caa gtg cgt gcc atc gag gag gcg ccg 432 Thr Arg Leu Pro Glu Tyr Leu Gln Val Arg Ala Ile Glu Glu Ala Pro 130 135 140 gta ccg acc gac caa tgc tgg gcc acg ctt gct cgg aca aaa atc gtg 480 Val Pro Thr Asp Gln Cys Trp Ala Thr Leu Ala Arg Thr Lys Ile Val 145 150 155 160 cag gag caa tcg gct gtt acg gtc gaa gcg gca ctc gcg atc gtt gct 528 Gln Glu Gln Ser Ala Val Thr Val Glu Ala Ala Leu Ala Ile Val Ala 165 170 175 cgc ctg ccg gag cta aag ggc att cga cgt ttc ctg gac ctc ggt tgc 576 Arg Leu Pro Glu Leu Lys Gly Ile Arg Arg Phe Leu Asp Leu Gly Cys 180 185 190 ggt ccc ggc atg gtg gcc att gcg ttg gca cgc gct ctt ccc gga tgc 624 Gly Pro Gly Met Val Ala Ile Ala Leu Ala Arg Ala Leu Pro Gly Cys 195 200 205 cat ggc act gcg ttc gag ttg ccg cca acc gca gca gtg gca cgg cag 672 His Gly Thr Ala Phe Glu Leu Pro Pro Thr Ala Ala Val Ala Arg Gln 210 215 220 aac gtc gag atg gcg caa ctc ggc acg cgt ctg tcg gtg ctg gga ggc 720 Asn Val Glu Met Ala Gln Leu Gly Thr Arg Leu Ser Val Leu Gly Gly 225 230 235 240 gac ctg acc cgt gat gaa att ggc agc ggt tat gac ctc atc tgg tgc 768 Asp Leu Thr Arg Asp Glu Ile Gly Ser Gly Tyr Asp Leu Ile Trp Cys 245 250 255 gct tcg gtt ttg cac ttc gtg ccg gat ctt gcg cag acg cta cgt aag 816 Ala Ser Val Leu His Phe Val Pro Asp Leu Ala Gln Thr Leu Arg Lys 260 265 270 atc cga gca gcg ctg gcg ccg ggc ggg gtt ttc gtg agc att cat gct 864 Ile Arg Ala Ala Leu Ala Pro Gly Gly Val Phe Val Ser Ile His Ala 275 280 285 gaa att ccg ctc acg gcg gcg cag aca gcc acg gtt ctg gcg tac tac 912 Glu Ile Pro Leu Thr Ala Ala Gln Thr Ala Thr Val Leu Ala Tyr Tyr 290 295 300 ctg ccg ttg ctg atg cgt ggc cac cat gtc tgg cac cag gga gaa ctc 960 Leu Pro Leu Leu Met Arg Gly His His Val Trp His Gln Gly Glu Leu 305 310 315 320 ccc gag gcg ttg ttc gcc gca gga ttc gct aac gtc gcc acc ttc gag 1008 Pro Glu Ala Leu Phe Ala Ala Gly Phe Ala Asn Val Ala Thr Phe Glu 325 330 335 agc gac ttg ttt cca ttg gcg cca gtg caa gtg ctg gtg tgc cga 1053 Ser Asp Leu Phe Pro Leu Ala Pro Val Gln Val Leu Val Cys Arg 340 345 350 <210> SEQ ID NO 6 <211> LENGTH: 351 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 6 Val Gly Asp Val Ala Leu Met Arg Ser Lys Met Lys Phe Gln His Ala 1 5 10 15 His Pro Leu Gln Ser Cys Trp Asp Leu Ala Leu Ala Ser Val Gln Ala 20 25 30 Asp Ala Leu Ala Thr Ala Leu Asp Ser Gly Leu Phe Asp Val Leu Thr 35 40 45 Glu Gln Ala Gly Ala Gly Lys Ile Ala Glu Gln Leu Glu Leu Asp Pro 50 55 60 Asn Arg Leu Glu Pro Val Leu Glu Leu Leu Trp Ser Met Asp Leu Leu 65 70 75 80 Glu Arg Phe Gln Leu His Asp Ser Thr Gly Ser Thr Pro His Tyr Arg 85 90 95 Ser Ser Asp Lys Ala Lys Arg Phe Phe Ala Lys Gln Ser Ser Asp Tyr 100 105 110 Cys Gly Asp Ala Trp Ala Phe Arg Leu Arg Ser Leu Arg Asp Phe Gly 115 120 125 Thr Arg Leu Pro Glu Tyr Leu Gln Val Arg Ala Ile Glu Glu Ala Pro 130 135 140 Val Pro Thr Asp Gln Cys Trp Ala Thr Leu Ala Arg Thr Lys Ile Val 145 150 155 160 Gln Glu Gln Ser Ala Val Thr Val Glu Ala Ala Leu Ala Ile Val Ala 165 170 175 Arg Leu Pro Glu Leu Lys Gly Ile Arg Arg Phe Leu Asp Leu Gly Cys 180 185 190 Gly Pro Gly Met Val Ala Ile Ala Leu Ala Arg Ala Leu Pro Gly Cys 195 200 205 His Gly Thr Ala Phe Glu Leu Pro Pro Thr Ala Ala Val Ala Arg Gln 210 215 220 Asn Val Glu Met Ala Gln Leu Gly Thr Arg Leu Ser Val Leu Gly Gly 225 230 235 240 Asp Leu Thr Arg Asp Glu Ile Gly Ser Gly Tyr Asp Leu Ile Trp Cys 245 250 255 Ala Ser Val Leu His Phe Val Pro Asp Leu Ala Gln Thr Leu Arg Lys 260 265 270 Ile Arg Ala Ala Leu Ala Pro Gly Gly Val Phe Val Ser Ile His Ala 275 280 285 Glu Ile Pro Leu Thr Ala Ala Gln Thr Ala Thr Val Leu Ala Tyr Tyr 290 295 300 Leu Pro Leu Leu Met Arg Gly His His Val Trp His Gln Gly Glu Leu 305 310 315 320 Pro Glu Ala Leu Phe Ala Ala Gly Phe Ala Asn Val Ala Thr Phe Glu 325 330 335 Ser Asp Leu Phe Pro Leu Ala Pro Val Gln Val Leu Val Cys Arg 340 345 350 <210> SEQ ID NO 7 <211> LENGTH: 705 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(705) <223> OTHER INFORMATION: ORF C <400> SEQUENCE: 7 atg tca cca gcc tgg aat tca ttc cag aag ggc tac acc acc atc tcc 48 Met Ser Pro Ala Trp Asn Ser Phe Gln Lys Gly Tyr Thr Thr Ile Ser 1 5 10 15 tcg ttc cag ggt att ccc ggt gag cgg cgc tac gag gcg gac gtt acg 96 Ser Phe Gln Gly Ile Pro Gly Glu Arg Arg Tyr Glu Ala Asp Val Thr 20 25 30 gtt tcg caa ctg cgc gtg gtg gcg tat gag ccg ctg atc tgc aaa tac 144 Val Ser Gln Leu Arg Val Val Ala Tyr Glu Pro Leu Ile Cys Lys Tyr 35 40 45 gtg ggc ccg gag cgg gcg gcg gag acg ctc ggt ggc aat cag ctc agc 192 Val Gly Pro Glu Arg Ala Ala Glu Thr Leu Gly Gly Asn Gln Leu Ser 50 55 60 cgt ctg gcg tat cgc gcc agc acg cca gcg aca atg gcc cat gcc aac 240 Arg Leu Ala Tyr Arg Ala Ser Thr Pro Ala Thr Met Ala His Ala Asn 65 70 75 80 gcg ttg atc agc cac ctg ttg cct tgt agt cgt ccg ctc tcg cgg ctg 288 Ala Leu Ile Ser His Leu Leu Pro Cys Ser Arg Pro Leu Ser Arg Leu 85 90 95 gac ctg cac atc cac acc ctt agc ctg ctc aac gaa cag ctc agc ctg 336 Asp Leu His Ile His Thr Leu Ser Leu Leu Asn Glu Gln Leu Ser Leu 100 105 110 ctg gcg ccg cag cac ggt gca gtt tca tcg ccg ttt tcg cca gcg gac 384 Leu Ala Pro Gln His Gly Ala Val Ser Ser Pro Phe Ser Pro Ala Asp 115 120 125 atc gac cgt atc gag cag gca cgg cag atg atg gat gag cac ctc gac 432 Ile Asp Arg Ile Glu Gln Ala Arg Gln Met Met Asp Glu His Leu Asp 130 135 140 aag ccc ctg acg ctc gac tat cta gcc acc atc gtc ggc atc aac aag 480 Lys Pro Leu Thr Leu Asp Tyr Leu Ala Thr Ile Val Gly Ile Asn Lys 145 150 155 160 aac aag ctc aag gac ggc atg atc tac ctg tac aac gcc acg ccg gcg 528 Asn Lys Leu Lys Asp Gly Met Ile Tyr Leu Tyr Asn Ala Thr Pro Ala 165 170 175 gag ctg ctg ctg gaa ttg cgc atg acc aag gct ctc gcg ctg ctg gaa 576 Glu Leu Leu Leu Glu Leu Arg Met Thr Lys Ala Leu Ala Leu Leu Glu 180 185 190 acc ggg ctg cgg gtt tcc cag gtc gcc tgg atg gta ggc tac aaa tac 624 Thr Gly Leu Arg Val Ser Gln Val Ala Trp Met Val Gly Tyr Lys Tyr 195 200 205 ccc aac aat ttc acc gtc gcc ttt act cgc tac cac ggc aaa tcg ccg 672 Pro Asn Asn Phe Thr Val Ala Phe Thr Arg Tyr His Gly Lys Ser Pro 210 215 220 aaa gcc ttg ttc ggc aag cga cta ctg gag ccg 705 Lys Ala Leu Phe Gly Lys Arg Leu Leu Glu Pro 225 230 235 <210> SEQ ID NO 8 <211> LENGTH: 235 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 8 Met Ser Pro Ala Trp Asn Ser Phe Gln Lys Gly Tyr Thr Thr Ile Ser 1 5 10 15 Ser Phe Gln Gly Ile Pro Gly Glu Arg Arg Tyr Glu Ala Asp Val Thr 20 25 30 Val Ser Gln Leu Arg Val Val Ala Tyr Glu Pro Leu Ile Cys Lys Tyr 35 40 45 Val Gly Pro Glu Arg Ala Ala Glu Thr Leu Gly Gly Asn Gln Leu Ser 50 55 60 Arg Leu Ala Tyr Arg Ala Ser Thr Pro Ala Thr Met Ala His Ala Asn 65 70 75 80 Ala Leu Ile Ser His Leu Leu Pro Cys Ser Arg Pro Leu Ser Arg Leu 85 90 95 Asp Leu His Ile His Thr Leu Ser Leu Leu Asn Glu Gln Leu Ser Leu 100 105 110 Leu Ala Pro Gln His Gly Ala Val Ser Ser Pro Phe Ser Pro Ala Asp 115 120 125 Ile Asp Arg Ile Glu Gln Ala Arg Gln Met Met Asp Glu His Leu Asp 130 135 140 Lys Pro Leu Thr Leu Asp Tyr Leu Ala Thr Ile Val Gly Ile Asn Lys 145 150 155 160 Asn Lys Leu Lys Asp Gly Met Ile Tyr Leu Tyr Asn Ala Thr Pro Ala 165 170 175 Glu Leu Leu Leu Glu Leu Arg Met Thr Lys Ala Leu Ala Leu Leu Glu 180 185 190 Thr Gly Leu Arg Val Ser Gln Val Ala Trp Met Val Gly Tyr Lys Tyr 195 200 205 Pro Asn Asn Phe Thr Val Ala Phe Thr Arg Tyr His Gly Lys Ser Pro 210 215 220 Lys Ala Leu Phe Gly Lys Arg Leu Leu Glu Pro 225 230 235 <210> SEQ ID NO 9 <211> LENGTH: 408 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(408) <223> OTHER INFORMATION: ORF G <400> SEQUENCE: 9 atg gca ttg ctt atc aag cgt cag gcg ctg ggg cag gtt ctg gct caa 48 Met Ala Leu Leu Ile Lys Arg Gln Ala Leu Gly Gln Val Leu Ala Gln 1 5 10 15 gca cgt cgc gat cac cca ctt gaa acc tgt gga atc gtg gcg tct tca 96 Ala Arg Arg Asp His Pro Leu Glu Thr Cys Gly Ile Val Ala Ser Ser 20 25 30 ctg gaa gcc cag tta gcg aca aga gta atc cca atg cgc aac cag gcg 144 Leu Glu Ala Gln Leu Ala Thr Arg Val Ile Pro Met Arg Asn Gln Ala 35 40 45 gca tca caa acc ttc ttt cgg ctc gac tcg cag gag caa ttc cag gtg 192 Ala Ser Gln Thr Phe Phe Arg Leu Asp Ser Gln Glu Gln Phe Gln Val 50 55 60 ttc cga tct ctg gat gat cgc aac gag ttc caa cgg gtc atc tac cac 240 Phe Arg Ser Leu Asp Asp Arg Asn Glu Phe Gln Arg Val Ile Tyr His 65 70 75 80 tct cat acc gcg agt gaa gcc tat ccg agc agg gag gac atc gag tat 288 Ser His Thr Ala Ser Glu Ala Tyr Pro Ser Arg Glu Asp Ile Glu Tyr 85 90 95 gcg ggc tat ccg gaa gcg cat cac ctg att gtg tcc aca tgg gag aac 336 Ala Gly Tyr Pro Glu Ala His His Leu Ile Val Ser Thr Trp Glu Asn 100 105 110 gcc cga gag ccc gcc cgt tgt ttc cgg ata ctt cgt gga aaa gtc atc 384 Ala Arg Glu Pro Ala Arg Cys Phe Arg Ile Leu Arg Gly Lys Val Ile 115 120 125 gaa gaa agt atc tcc att gtg gaa 408 Glu Glu Ser Ile Ser Ile Val Glu 130 135 <210> SEQ ID NO 10 <211> LENGTH: 136 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 10 Met Ala Leu Leu Ile Lys Arg Gln Ala Leu Gly Gln Val Leu Ala Gln 1 5 10 15 Ala Arg Arg Asp His Pro Leu Glu Thr Cys Gly Ile Val Ala Ser Ser 20 25 30 Leu Glu Ala Gln Leu Ala Thr Arg Val Ile Pro Met Arg Asn Gln Ala 35 40 45 Ala Ser Gln Thr Phe Phe Arg Leu Asp Ser Gln Glu Gln Phe Gln Val 50 55 60 Phe Arg Ser Leu Asp Asp Arg Asn Glu Phe Gln Arg Val Ile Tyr His 65 70 75 80 Ser His Thr Ala Ser Glu Ala Tyr Pro Ser Arg Glu Asp Ile Glu Tyr 85 90 95 Ala Gly Tyr Pro Glu Ala His His Leu Ile Val Ser Thr Trp Glu Asn 100 105 110 Ala Arg Glu Pro Ala Arg Cys Phe Arg Ile Leu Arg Gly Lys Val Ile 115 120 125 Glu Glu Ser Ile Ser Ile Val Glu 130 135 <210> SEQ ID NO 11 <211> LENGTH: 270 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(270) <223> OTHER INFORMATION: ORF H <400> SEQUENCE: 11 atg tcg att tca gtg atc gtt ccc aca ttg ctg cgc ccg ctg acc aat 48 Met Ser Ile Ser Val Ile Val Pro Thr Leu Leu Arg Pro Leu Thr Asn 1 5 10 15 ggg gaa aag aca gtt ttt acc caa ggc aac tcg gtg gca gag gcc atc 96 Gly Glu Lys Thr Val Phe Thr Gln Gly Asn Ser Val Ala Glu Ala Ile 20 25 30 gag aac ctt gaa cac cag ttc cct ggc ctt aag gcc cgg ctg gtc agt 144 Glu Asn Leu Glu His Gln Phe Pro Gly Leu Lys Ala Arg Leu Val Ser 35 40 45 gcg gaa cat gtg cat cgt ttc gtc aat atc tac gtc aac gaa gac gac 192 Ala Glu His Val His Arg Phe Val Asn Ile Tyr Val Asn Glu Asp Asp 50 55 60 atc cgc ttc tca gat ggg ctc aac acg cca ctc aag gcc ggt gac agt 240 Ile Arg Phe Ser Asp Gly Leu Asn Thr Pro Leu Lys Ala Gly Asp Ser 65 70 75 80 ttg acc gtg ctg cct gcc gtc gcc ggt ggc 270 Leu Thr Val Leu Pro Ala Val Ala Gly Gly 85 90 <210> SEQ ID NO 12 <211> LENGTH: 90 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 12 Met Ser Ile Ser Val Ile Val Pro Thr Leu Leu Arg Pro Leu Thr Asn 1 5 10 15 Gly Glu Lys Thr Val Phe Thr Gln Gly Asn Ser Val Ala Glu Ala Ile 20 25 30 Glu Asn Leu Glu His Gln Phe Pro Gly Leu Lys Ala Arg Leu Val Ser 35 40 45 Ala Glu His Val His Arg Phe Val Asn Ile Tyr Val Asn Glu Asp Asp 50 55 60 Ile Arg Phe Ser Asp Gly Leu Asn Thr Pro Leu Lys Ala Gly Asp Ser 65 70 75 80 Leu Thr Val Leu Pro Ala Val Ala Gly Gly 85 90 <210> SEQ ID NO 13 <211> LENGTH: 25801 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 8300,8319,8338,8708,11735,11768,18459,18686,18703,18711, 18720,19226,20077,20506,20545 <223> OTHER INFORMATION: N = unknown nucleic acid residue <400> SEQUENCE: 13 attcccgggg atctgatcat ctgcgaggga cgcaatctgc acccggagga catcgagcac 60 actgtgatcg aggcgctgag cgacttgcgg gcgcaaagct gcgcagtgtt cagccatgac 120 gacgaccagc agcgccagac catcgtcgca gccatcgaac tgaatcgtga actcaagcgc 180 cgcctgcagg acaattgccg ccagctcaag gccgtggtgc gtagcgcggt ggtggacagt 240 catggcatca ccctcaaccg catcgtcttc gtgcagccga ccagcattca caagacgacc 300 agcggcaaaa tccagcgcgc gaagatgcgc cagctctatc tcgccgagga actggacatg 360 ctgcaatgaa acccggtgcc ccttcagccc tggtgcaagt ggctggccga tgcggcggca 420 ccagtgagcc tggtctgttt ccattgcgcc ggcggcaagc cgcscaarak gtttttcccc 480 gtggaaaaaa ggccgcccaa ggcctttgcg agctgtatgc cgtctgaact gcccggccgc 540 agctcggcgc ttgcgcgagc cgttcgccga gtcgctggcg caactggccg aggccttcgc 600 cgagcagtgt cgcgccctgc cgaacaaacc gcttatcctc ttcggtcata gcctgggcgc 660 actgctcgca tatgaaaccg ctcgtgtgtt gctggccaaa ggcgaaaggc cacccgtgca 720 gctgctggtg tcctcgcgcc agagcccgga ctgkctgccg gcctgcgcgg gtctgccggc 780 gctaaacgat caggctctgc gcgattacct aggcaacctc gccggcaccc cgccagaggt 840 gctgcagagc aaggcgatga tggatctcgc ggtgccggtg ctgcaggccg acctgaagct 900 gatcctcaat taccagcatc gacatctcca gcccctgagt atcccggtgc tggtcttcgg 960 cgcgatcagc gaccaacagg tgcgctatga gtcgctgctg agttgggagc ggatcagcgg 1020 cgaggggttc agcctgcgga tgatcgaggg agggcacttt gcggtaatgc agcaaccgca 1080 gtgggtgctt gaccaggtgc aaaccgagtt atagccctcc cagccagctc ccatacgcag 1140 gcacatctct gtttgcgcct cgctcttcgc cctctcgcgt tggacataak raactcgccc 1200 atgcgcgggg gcaaaagata tcccaggtcc ttgacgtgtc gtgaatcgcg atccgatttg 1260 ccgtcgaggc tttttgccgt gttctcgagg cgagtagaag cgcaaccggg gcttaaactt 1320 cattcgccty gccagacggg cccccgcttg cacttcatga acttgcaaac ctgacatgca 1380 agggtgcacg cctgcgtttt acccaccaat ggcatgccct tatcaatacc aatccgaagg 1440 tactggcgtg cgcgtgctcg tarctgataa gaacgtccat cccaacaagc cgtaaaaacg 1500 gtcaaactgc gtttttgggc aatttaatcg ccgcgatgct ctagcagggg cagcacaatt 1560 gggagcactc gaggacttcg atcccgaacc ttcgcccggc tggacaaagt gacacaagcc 1620 gatgctagcc ccacggttga aacccgtatt cgctgctacc ccgagcagcc cggcaattat 1680 ctgctgcgtt ggtcgttcaa atcctgtgaa actgatccta cgaaagccgg tatcggcata 1740 tcgcaaggcc tctggggcaa attttctatt tcttgaccgc ccccaaccgg ataccaaaag 1800 cttcgcgttt accgcgggtt ttatcgagtc tggacggcgt raaagggcag gcttgccctg 1860 gacttggtgc cggactacta cattggtatg cagcgcctgg cactttttgc atatttcgcc 1920 atagcgacgg ggttcctatg ctgacggcaa ctccacagga gtcgcccatg tcatttcatc 1980 acctgagagc gggttgcctg cccttgactg cttcgcagct ttggcgaacg ccacaccaac 2040 cgatccagtg ctgggcgtga agatcggcta tctggcttat cgaccgaacg cggggccatt 2100 gctctccaac gtcctcagcg aggcgaaacc atgatggcaa aacatttccc tctactaagt 2160 ttggtcctcg cgataccagt tacgcacgct cagaccagtg atctgagcta tgagagcttt 2220 gtggatagcg atagacgcat caaatgcctc tacggctatg ccgctgaaaa gacgggtgat 2280 cacgagtcgg cgatcaagat tttcgaagat tgtgtcgagc gctggaacga tgtgtattcg 2340 atgatttggc ttgcccagat gtacgaatcc ggcgcaggtg tggatgtaga cctggcgaaa 2400 gccgcatcgc tcatgagwac gcggcgcaga gcggcccgac gatgttgcgt acgtcagtct 2460 ggcacgctat cactatgggc tcgcgctggc ggaagggcga ggagtcagaa aagaccttca 2520 agccgctcgt gcctggttgc aaagcgcctc aaaaggtggc cagagcgaag cgaatgacta 2580 cctcacgcaa ctggaaaagt catcctcttt gaacgctccg gctcgtcaac accctagcgg 2640 agcsgaataa acgctttgcc agctcctacc gagcgcgtca ggtgtatgac aacgtgctgc 2700 acaagcacag caagtcccag gctcgcttcg gtcgctcggg cctagacatg ctggcgtacg 2760 accctctgga agaagggccc ttgtacctgt tcggtgtgag cgcgagcgcc caagctaacg 2820 gtgagcttca ctacgacatc tgagcttttt tcggagttag gtgatgcgct gaacctggtc 2880 gaactccatg gccgcattta caaccaacct ctgcgcataa cgacgacatt tatgtggtga 2940 tgatcagcgt gcgctcgaga tcctcacttt ctcaggtgga acagggcgtg aagcgaaaat 3000 cgacatcaaa tacacagtca agcccatatc gcgaacgacc ttcttccgaa tgctccgacg 3060 caagaccgaa taggctctgg tagatgtgcg ccgatgctga cgctgagtag aaggatatcg 3120 agacagcaga gatggactac ccagaggtac ctacggtttt atccgagcat ttacaaggcc 3180 ggagcccgat agccgctgat tatgaagttt gacaaccgac cgcttttggc gaggtcgccg 3240 acgatacgcc gcacaggccg ccagcccagt acatgatttt cgccgctgtc gttctcgaca 3300 gcggcgggag ggatgcatca ttcagtacat taggctggag cgcccgtcat cgatcacggg 3360 cgcaccgagg tagcttacgg ctccagtagt cgcttgccga acaaggcttt cggcgatttg 3420 ccgtggtagc gagtaaaggc gacggtgaaa ttgttggggg tatttgtagc ctaccatcca 3480 ggcgacttgg gaaaacccgc aagcccggtt ttccagcaag cgcgagagcc ttggttcatg 3540 cgcaattcca gcagcagctc cgccgggcgt ggcgttgtac aggtagatca tgccgtcctt 3600 gagcttgttc ttgttgatgc cgacgatggt ggctagatag tcgagcgtca ggggcttgtc 3660 gaggtgctca tccatcatct gccgtgcctg ctcgatacgg tcgatgtccg ctggcgaaaa 3720 cggcgatgaa actgcaccgt gctgcggcgc cagcaggctg agctgttcgt tgagcaggct 3780 aagggtgtgg atgtgcaggt ccagccgcga gagcggacga ctacaaggca acaggtggct 3840 gatcaacgcg ttggcatggg ccattgtcgc tggcgtgctg gcgcgatacg ccagacggct 3900 gagctgattg ccaccgagcg tctccgccgc ccgctccggg cccacgtatt tgcagatcag 3960 cggctcatac gccaccacgc gcagttgcga aaccgtaacg tccgcctcgt agcgccgctc 4020 accgggaata ccctggaacg aggagatggt ggtgtagccc ttctggaatt ccaggctggt 4080 gacatcctgc ccgtcgtagc aggagcggcc tttcatccca acggtgatga ccatgacccg 4140 accttcatgc ggtccgctcg tttcctcgat cagcggcaca gtcggacgat agtgcagcct 4200 gccaagaccg aggcctggtt caagttcgac aaaatcctgg aagcattggc caatctcgtc 4260 gggcagctgc agccgagtct tgccatcgct caggacgctg cggatgcgtg gttcttccat 4320 tgtcagccgg tagcagctct tgtgtgcgct cacctctcac ctccagtccg ttagcccccc 4380 gaatgtcgcg catatcaatc tgtgtttctc gcatacaaaa aatgctaatg gttctcatta 4440 ctataatgca tcattttacc gatggccagt cattggcgac caaacaccca actaaaaata 4500 cggtaaaatc aatgtcttac aggccaggca ctgcacgccc gagaggaact ggacgactac 4560 ccacgaagcc aaaccctgtc gaaacactgg cgttcccttc tctccttgct cgaccgcacg 4620 cgctgcaatc gtgttggccg acgcagctga ccgagccgct gcgcgatggc aggccgtgtc 4680 cggtctcacc cgctcctgct ggacggcggc agggatgggc cgataccgcc cgggtgaaga 4740 tggctcaggt gcaggcggtc agggccgacg tgtcctgtga gattcttcct cgacagcccg 4800 tgttttcgcc gataggacgg acactgaaca tggacggcat gcccggcttg gtggcggtcg 4860 cactggcgaa gccgaagctg gaagtgtccg gtgtagggtt cgagtgcccg gccgccgccg 4920 tcgtggcgca gcagcccatc gaaagcgtcg gacttcccga ccgtctgagt gcgcgcagcg 4980 gcgatctggt acaggaggtc gagcaacggg atgacgtctg ttttccgttc gcttccgtcg 5040 ctggcctgct cgcccgcaaa gaggaaaacc tctcatgacc aagtcgcgag tggtaggtct 5100 gcaactgttg ttcggctgga tgaatctggt gctggcggta cccagcatct acttgatgct 5160 cggcatgcca cttgtaatgc gccagcatgg ctgragcggc gcagagatcg ggttgttcca 5220 gcttgccgcg ctgccggcga tattcaaatt cctgttggct gtgccggtgc agcgtgtgcg 5280 cctcgggcgc ggacatttcg tgcactggtt gctgttgctc tgtgcgctac tactggcgct 5340 gtactggcta atcggacggc ataatctgat cggcgatcgc ataatgctgt tcgcgctgac 5400 cttcgccatc agcattgccg ccacgtgggc cgacattccg ctaaatgcgc tagcggtgca 5460 gtggttgccg cgtagtgaac agttgcgcgc cggcagcatc cgttccgcag cgctgttcgt 5520 aggcgccatt gttggcggcg gcgtcatgat catggtgcag gcgcgcgtgg gctggcaggc 5580 ccccttctgg ctgctagggg tcggactgct gattggcgcc ctgcccttcc tgctgttgcg 5640 tagacacgcc gcactgcccg agcaggccga gccgcgcgag actacagatc ctccaccggg 5700 cgtgatggcg gactgggcaa gcttcttcca ccagccaggg gcgcggcaat ggacattgct 5760 gctgctgacc agtttcccct tcctcggcgc gacgtggctg tacctcaaac ctttattgtt 5820 ggacatgggc atgcagctag agcgcgtggc cttcatcgtt ggcatcgtcg gcggcaccgc 5880 aggcgcactg ttcagcctgc tcggcggaca gctagtgcaa atgttgggca tagcacgggc 5940 cattgcctgg tacctgctgg cggcgctggg cgcgctggca cttttgacgt tcagcgtctg 6000 ggcccaactg ggggcggcat ggctgattgc cagcgccctc tgcgtggcag ccagcatggg 6060 cgccatctcg gcgctgatgt tcgggttgac catgttcttc acccgaaatc ggcgcaacgc 6120 gtcggactat gccctgcaaa ccaccatgtt caccgttgcg cgactggcgg tgccgatcgc 6180 cgccggggtg ttgctcgacc gggtgggcta caccggcatg ctcctggcaa tgaccctggc 6240 gcttctgctt tccttcgcgc tcgcctgtcg ggtgcgggaa aaggtggaat cttcggcaca 6300 gtcgatactc gagcacgagc gggtttgaag gctgaagtga ccggccatgc cccttcggac 6360 aatggcctga aatgcgcggt ccttttgcat agtttttcat gctcacgtca tatgaaagaa 6420 cagccaacgg caattgctat agtcatcacc acgaacgata atgattatcg ttaccattga 6480 aatcaaacag gataagcgat atgccactat cagcgctggt ggcgccggct ggggaactga 6540 gccgcgccga gatcaaccgt tacagccgcc acctactgat acccgatgtg ggcatgatcg 6600 ggcagcgtcg gttgaagaac gccaaggtgt tggtcatcgg cgccggcggt cttggctctc 6660 cgactctgct ctatctagct gcagcaggtg tgggcaccat cgggataatc gactttgatc 6720 gggttgacga ctccaacctt cagcgccagg tcatccacgg ggtggatacc gtgggcgagc 6780 tcaaggtgga cagtgcaaag aaagccattg cgcgactaaa tccctttgtc caggtcgaaa 6840 cctataccga tcgcctggaa cgggacatgg cgatcgagct gttttcgcgc tacgacctga 6900 tcatggacgg taccgacaac ttcgcaaccc gttacttggt caacgacgcc tgcgtgctgg 6960 ccaacaaacc ctatgtgtgg ggctcgatat tccgtttcga agggcaggcg tccgtgttct 7020 gggaaaacgc cccgaacgac ctgggcctga actaccgcga cctgtatccg gagcctccgc 7080 cgcccgagat ggccccctcg tgctccgagg gcggtgtgtt cggcattctt tgcgcatcca 7140 tcgcatcgat catggccacc gaggcggtca agctgatcac gggcatcggc gagccactac 7200 tgggtcggct ggtggtgtac gacgctttgg atatgcgcta tcgggagctt cctgtgcgcc 7260 gcctgccaaa tcgacaaccg atcaccgacc tggccgagga ctatcaggtg ttctgcggtc 7320 tggggttgcc caaaggtgac acggcggacg ccgtgccagg gatcagcgta acggaactca 7380 agaagcggat ggatcaggac gaggtgcctg tgctgataga cgtgcgcgag cccaccgagt 7440 gggacatcgt ccgtattccg ggcgcaatct tggtgaccaa atcgcccacc gcagcgcaga 7500 cactgcgcga gcgatacggg gcagatgcca acctggtgat cgtctgcaag tccgggcggc 7560 gctctgccga cgtgaccgcc gagttgytaa acctgggcat gcgcaatgtt cgcaacctcg 7620 aaggtggcgt tttggcctgg gtgaaggacg tggactcttc tctgcctagc tactgatagg 7680 aggcctagaa gatggcattg cttatcaagc gtcaggcgct ggggcaggtt ctggctcaag 7740 cacgtcgcga tcacccactt gaaacctgtg gaatcgtggc gtcttcactg gaagcccagt 7800 tagcgacaag agtaatccca atgcgcaacc aggcggcatc acaaaccttc tttcggctcg 7860 actcgcagga gcaattccag gtgttccgat ctctggatga tcgcaacgag ttccaacggg 7920 tcatctacca ctctcatacc gcgagtgaag cctatccgag cagggaggac atcgagtatg 7980 cgggctatcc ggaagcgcat cacctgattg tgtccacatg ggagaacgcc cgagagcccg 8040 cccgttgttt ccggatactt cgtggaaaag tcatcgaaga aagtatctcc attgtggaat 8100 agcgactttc caatatttca atcagcaatg cctcagccac agctagaggc aaaaggagtt 8160 ctacatgtcg atttcagtga tcgttcccac attgctgcgc ccgctgacca atggggaaaa 8220 gmcagttttt acccaaggca actcggtggc agaggccatc gagaaccttg aacaccagtt 8280 ccctggcctt aaggcccggn tggtcagtgc ggaacatgng catcgtttcg tcaatatnta 8340 cgtcaacgaa gacgacatcc gctttctcag atgggctcaa cacgccactc aaggccggtg 8400 acagtttgac cgtgctgcct gccgtcgccg gtggctgact cgcacctccg gacaccgctg 8460 aaagaatgac ccctggcgat tcaaatccag gggcaagtgc aacgcttttg tctctcggtt 8520 ttgaatatgc cgacactctt aaatgaattt tccctgctgc attcatccac ttcgtttccg 8580 ccgaattgga atgaactgca acttagcctg acggaacagg ccagattatt gggcatttgc 8640 ccgctcgcaa tctcgccgcc tgtgratatg gaaggagccg cattccagct gcagcatccg 8700 gctatttntc ctattcaggc ccacttcgcc tcaccagccg gctggctgcc aaatcgacac 8760 ctctcggagc tgctgctgca ggcgggcagc ggtcttatgt cggtgcacgg ccgtgctagc 8820 ggtagggccc aaccgctggg cgtggattat ctttcgacac ttaccgccgt catgacgctg 8880 cacggaacgc tggccgcagc cgtggggcag ctgcgtggcg gtgcatttga tcaggttcag 8940 ctttctccac tgggatgcgg gctgctcagt atcgggcagt atctggcagg cgccacggca 9000 ccagaagatc gtgaggcgtt cctgccgggc ggctccgatc cgcatttgag gccgccattt 9060 cgttccgctg acggcatcac attcgagctg gaaacgctcg acagcacacc gtggcgaagc 9120 ttttggaccg ccgtcggcat tgaatcggaa ttggccggta cggcctggaa aggttttctg 9180 cttcgctacg cgagggccgt gtcgcctcta cctgccgcct gtctcacggc gctcgcccgc 9240 ctgcgttacg caaagatcca acaattggca gcgcaagcgg gtgttgcggt cgtgcccgtc 9300 cgcaccgatg cgcaacgccg cgaggacccc gattaccggc agtcactggc tacgccatgg 9360 cagttcgagt ctttcccgcc gtcccccgaa aggcatcgag acaccgcatt tccgtcactg 9420 ctgccgctac aggggatgcg cgtcatcgaa tcctgtcgac gcattcaggg accgctggcc 9480 gggcatctgc tggcatcgct gggcgccgaa rtcattcggc tggarccgcc gggtggcgat 9540 ccgttgcgar ccatgccgcc ctgcgccgaa ggctgttcgg tgcgctttga cgcgctgaac 9600 cacctcaaat ccgttcacga agtcgatatc aaatccgccc atgggcggca gttggtctac 9660 gagctcgccc gcgatgcgga tgtctttctg cacaactggg cgcccggcaa ggcccatgaa 9720 atgcaactgg atgctgaaca tctgcgcagg gttcaaccac atctcgttta cgcctatgcg 9780 ggaggctggg gccgggctcc cgtcaatgcc ccgggtaccg acttcaccgt ccaggcctgg 9840 tcgggtgtgt ccgccgccat tgcacgtcaa tccggcatcc gcggcggctc gctgttcacc 9900 gtgttggatg tgctgggcgg cgcgatcgcg gcactgggtg tgacggccgc gttgctcaat 9960 cgagcagtca cgggcacggg tacttatgtc gagagctcat tgctgggcgc cgccgatctg 10020 ctgatgcaca gcagcggcaa ggcgtcgagg ggcatcttgt ccggcgtgta tcccacgcta 10080 tcgggactga tcgccatcga ctgccaacac ccagatcagt tccagtcgct ggccatgttg 10140 ctggacattc ctgccactgc gratacctgc cagcagacgc tgcggagcgc ttacgcaagc 10200 gacccgcttc ggaatgggaa acggtgctga acgaacgggg catcggcgcc tgtgtagtca 10260 tcgaagacct caagcagctc gccgccgaca cccgcatctc tgaatgcctc actcgcaagt 10320 cttacttctc tgtcaacgcc ccctggaggt tcctatgaac aacgctggca tcatcgacct 10380 ggttcctgct gaggaacgcc aacgttgggt gcaggacggt acctacccga accagcccgt 10440 attcacgctg tttgccgcca aagccgaagc gcatcccgac aagaaggccg tgctgtcgcc 10500 gcaaggtgac gtgacctacg gcgagctcct cgatgcagcc ctgcggatgg ctcacagcct 10560 gcgtgattcg gggatcgtgg ccggcgacgt ggtggcttac cagctcacca accactggtt 10620 gtgctgcgca atcgacctgg cagtggcagc gctcggtgcc atcgtcgccc ccttccctcc 10680 gggacgcggc aagctggata tccaatcgct ggttcgccgc tgcgacgcgc gagcggtgat 10740 cgtcccgcaa gcgtacgaag gcatcgatct gtgcgaggtt atcgagtcac tgcgccccac 10800 cctgctatcc atgcgccgcc tgattgttca gggcaagcct cgcgaaggat ggattacgct 10860 cgatgagctg atgagcaccg agccgctgga tctcgccagc ctacccaggg tgtgcccgaa 10920 ctcgccggtg cgtctgctgg tgtcttcagg caccgagtcg ragcccaagc tggtggcgta 10980 ctcgcacaat gcgttggttg gtggtcgcgg gcgcttcctg cagcgcatcg cgtccgatgg 11040 cgaagatttt cgcggcatgt acctcgttcc gctgggttcg tccttcggct ccactgccac 11100 cttcggtgtg ttgtgctggc tgggtggttc gctggtcgta ttgcccaagt tcgacgtgga 11160 tgaagccatc aaggcgattg cggcatttcg gccgggcttc attctcggcg tacccaccat 11220 gctgcaacgc atcgccgctc aaccggcgtt ggagagcatc gacaaatcca gcctgcgtgg 11280 tttgatcgtc ggcggctcgg tcatcgacga ggccaccgtg cgcaaatgcc gtgatgcgtt 11340 tggctgcggc ttcatcagcc tctacggttc cgccgacggc gtgaactgcc ataacaccct 11400 ggacgacccc atcgaagttg tgctgaccag cgtcggcaag cccaatccgg cggtctgcgc 11460 gattcgtctg gtggacgacg aaggccggga ggtccggcaa ggcgaggttg gcgaaatcac 11520 cgcccgcggg ccattgactc caatgcagta cgtcaacgcg ccggagctgg acgagcgtta 11580 ccgcgacccg caaggctggg tgaagaccgg agatctgggc tacatcaacg acaagggtta 11640 tctggtccta gccggtcgca agaaggacgt catcatccgt gggggcgcca atatcagccc 11700 gacccagatt gaaggcctgg tcatggcgca tcccnatgtc gtgaccgttg cgtgcattcc 11760 tgttcccnat gatgatctcg ggcagcgggt gtgcctttgc gtcacctttg cgcgagggtg 11820 cagcgaagtt ttccctgaaa gcgatcaccg acttcctgcg cgaactggga ctggaggtga 11880 acaagctccc cgagtaccta cgcttctacc gcgctctgcc tctgacaccg gcgggaaaga 11940 tcgataaaaa agcgctaacc gaggaagccc gcgagctggg cacctcgggc atttgtcccg 12000 ctgggccggg gcagtcgact cccgagcgca gcttacggga gtacgcatga tatgcgcggt 12060 caaccgatga tgatggctac agctttgatc tgtgcctttg taccagggcc acagttggcg 12120 tttgctgcgc caggctccgc ggcttcgcct gactccacga cgctaccgga aatcaccgtc 12180 acagccgaga aaatcgagcg gccgctggaa agggtgcccg ccagcgtggc ggtgatcgat 12240 ggctgggacg ccgagcagtc aggcatcact agcctcaaac aactggaagg acgcattcct 12300 ggtctgtcat tccagccgtt cgggcaagca ggtatgaatt cacccgtcat gcgggggctg 12360 acggccaact tcaacagctt ctccagttca acgttgttgc tggtcgatgg cgttcccacg 12420 ctgacagccc aggggattcg agagtggcat gctggatctc gatcgcatcg aggtcattcg 12480 cggcccgcaa tctacgctgt atggccgtaa tgccgaggcc ggtgtgattg ccatccacag 12540 cctgccgatg gacgcgaccc cgagagccag cgtgtctgcc gaagcgggca gccggaacaa 12600 gcgtgtcatg cggtttgcgc tcagccagcc tttggtggaa garcggttgt acggcagcgt 12660 atcgggcaac tggtcgagcc aggacggctt catcgacaac acccacacgg ggcacaaggc 12720 ggacgatcgt garcagaaga acctgaacct ggggctgcgc tgggccccgg gggccgcaac 12780 ggatgtggtc atgcgctatg cgcatcagga gtacaacaat ggcgcctccc tgtggggctc 12840 gcccggcgcg ccaaggaaac aagtcccgtc cggaacgccc aactggaacc gttctgaggg 12900 ccagaccttg tccttcaatg tycagcatga atttgcctcc ggctgccgtt gcattcggta 12960 acggcctgga acgattcaag gacaggattc agcaggacac tgacttcatg ccagccgatg 13020 ttctgcacgt cgggcgcgac catcacctgc gcacactctc ccaggagttc cgtgtggagg 13080 gacagctcgg ggaggccagt tggctggctg gtgtctacgc ggatcgcagc gacaacgatc 13140 tgcacagtac cagcaagacc atgatggggc tgtcggacat tcgcgcggat cagcagagcg 13200 atacgctgca ctgttcaccc actggaacgt ccccctgtcg gccgactggt ccatagacgc 13260 cggagcgcgc gtcgagcgca acgaggtgca gctacgtccg caaggggcta cgagccatga 13320 aaaaggcwgg acacacgttt cacccaggct cgcgctgcaa caccagataa ccgccaatca 13380 ccaatggtat gtgagtgcca gtcgtggcgt gcgcactggc ggcttcaatg tgctggcgcc 13440 gacgctgggt tatctgcctt acgacacgga gaaraactgg tcgtatgaaa ccggtctcaa 13500 gggctggctt cttgacaagc gcattcgcta ttcgctggcc gcctacctca tggacatcga 13560 tgacatgcag gtcatgcaga tgcccaccgt cggcgtgatg tacatcacca gcgctgccac 13620 ggcgacatcc aaaggtctcg agctggatgt ggactatctc ctcggtggcg gctggcagct 13680 caagggcggg ctggcctgga aaccacacgc cgcttcgatc actttcgcga tggcgaggcg 13740 gactatgacg gcaaccagaa cccgtttcgc gccggatctc accggccacc tcggcatccg 13800 ctacgacgcg cccgaaggct ggtatgcaca agccagcgtg accggcagca gcaaggtyta 13860 cytcgatgcg gccaacgggt atgaacgcaa cggctacggc ytggtgaacy tggtagttgg 13920 ttaccaacgc ggcaactggg aaatcgcggc ctacgccgac aacgcgaccg atcagcgtta 13980 cgacgcggtg ggctaccaga acggattcgt caccgtctac agcccgccgc gagaagcggg 14040 cctgcgtctg acatggcgcc tgtgaacaaa gtcgcyggaa tccgcgggat cgggcaatgg 14100 cagcaagcca aagaggatgc gggcatatgc agcaagagag acggaatatc ggaatactct 14160 tggtcagcca ggatgaaaaa ctggcgttgg acctggacat ggtagtggaa agcgtaaacg 14220 gtttgctcag tcgcaatacc gatacgcctt tcgatttgca cccgaacgac gaatgctttc 14280 cctaccgcca gatcttcgct cagrcctgtc gatacttgcg ctcacaaccc agggacaagc 14340 tgcccgccct gttctctcgg tgcttccact caatggcaac cgcccgccag gcgctgtcgg 14400 cgggtgattg gacgttgtcc ccgcttgaat gcgtgttgct cgatacgcgc catgaggaac 14460 cggtcgatga cgaccccttc cttgcgctta cgctgcaaca accgccgacc cggccctcgc 14520 cctgctctgc aatgctgctg tgcgaggaaa gggtgctggg taaatggatg tgccggctgg 14580 ggggcaaccg cctggtgcgg gtgccgcatt cgaatccctg gcaacgccgc gccgatatyc 14640 tccsggctga ttctggatca tctggagcat gcccatttca acccgcatgc ttgctcgcsc 14700 gcgccagaaa gccgatggcc aggtgacgct ggcacaaaag attcatcacc tcatgactga 14760 acgctggggg tgatcaatgg gacttccact tctacacggg ctccatggtc gccggtttca 14820 tcgactcgat gaaatctctg ctgcagggaa cggacagcca ctgcctgacc ggtaacaacg 14880 agcactcatt ggccgtaagc gcgctggctg gctggcagtt gtatgrycgt gcctacgttc 14940 atcgccattg actccgggaa tgatcgacga agcgcgcggg aactctggcg aatctcaagc 15000 gtgccgcaag cccctggcat cattgtctgc gccgactcmc ccggaaacga tctggtatcc 15060 attccagggt acgctcgacg ccgacagcga cggacatgcg gtcattgcag cacgcggctt 15120 gtggcatggg ttcatgcgta cccccgatga catgccagct tgccttgcaa atgccttcca 15180 ggcccttgat gagcgccccg ctccaacctt cgtccttgca acgcaacacg tgctggagtc 15240 gcaaagcgac catttcggaa ccagtggtgc agcgcccctc ctccaaggct gcaacgctct 15300 cctgcgcgca acgcgaacga ctggatcagg ccgtggccgc catcaaccac gacaacgccc 15360 ggatgctgtg gcattgcggt cgcctcacat cggacgagcg gaatcagatt cttcgcctgg 15420 ccgaaaaagc cggcatcgcc ctggtggaca gcatcattca tccgggaagc gtgcccgggt 15480 tcagcgacgg caagtccgta gcgaactatc tgggaacgct ttccatgtat ggattcaacc 15540 gcgcggtcta tgaattcctg gaagcgcaaa gcaccaacga agaaggcgct ccctggctct 15600 ttttcctcaa gggcaaggta gagcaatcgt ctaccccgta ttcggaaggc aagctcaagc 15660 gcaacttccg cattggccaa gtcaactgca atgaggcgca cctgtcgcca ttcaccctgc 15720 tgggactgga cgttcggttg gcggacttcc tgaattacct cgaaccacgc ctgcaggttg 15780 acgacgcggt gctacggcag cgccgcgcca gaatcgaaca attgcgcaag ctgccggccg 15840 ctcagccaag cgacctgatc gaaacaatgc cgatgacccc gaattatttc ttccaccagt 15900 tgggacgact ggtggtcgat ctgatcgagt cgcgaagcta ccgctatacc ggcgtctacg 15960 acgttggtcg ttgcggtttg tcggcaatgc gcaacgtggc acgcaccgat cccggtttct 16020 ccggctggta cggtcgcgcc ctgatgggtg acggtctgat gtcgctgccc tatatcgcct 16080 tgaagaacga acgcaatgtg ctggcattca tcggtgatgg tgctcgcgca atagtgccga 16140 atgtggagca gcgcctggtt ggttctttcg atccggggca tcgcagggcg tggtggcaac 16200 gtcaccgttt tctatctgag caacggcgtg ctttccatga ttcagaccta tctggacaaa 16260 cgctacacgc tcaatggctg cagccaggtc aacgtgccat tgacggaatg gaaggaggct 16320 cccgtcgagc atgctgaaga cgatgtgacg gttcatcgtc gcgttatccg gcaattctgc 16380 ccggcgctgt tgggcgaggc cttgatggct ccgcgccgcg tcaatttctt tgatgtctgg 16440 cttggacaca actcggaggg cgatggcttg agcctgatct cggaagggtc ctggagccgc 16500 atgcgtactc atggagagtg aacacattat gaagttcggt tttattgccc accccacatc 16560 ccgggacttg ctgcatcagg tcaagctgat cgatatggcg ggtcgtatgc tcgaggaaca 16620 ggccaacggc tacgacggcg agcgctggcg ccgtcgcaat ctggttccgt tcattgaatt 16680 cactcgcatc gtgagcgcca gcggttcgca gtgtgaaggc atcttgcagt tcatgccgtt 16740 gacgcagagc aaatgttgag tcagccgagg cgcattgcgg agcgtgtcgt cgaaggcgtc 16800 aatgcgttaa aggacgaggg ggcagaactc gtagggctgg gaggcttcac gtccattgtt 16860 ggcaatcgcg gcctgcagac actggatcgc acgcaaattc cggtgacgac gggtaactcg 16920 ctgactgcgt atgccgccta tatgaatctg ttgggtgtcc tatccgcgct ggaaattcca 16980 ccagaaaaag cggaggtcgc cgtgctcggg tatcccgggt cgattgcact ggccatcgtt 17040 tgcctgctgg cgccattggg gtgtcggctg cgactggtgc atcgcggagg gaaacagcag 17100 gtcgacacgt tgcttgagta cctgccctca caatttcacg gacaagtgac gctgcacgcc 17160 ggtctggaag attgctatgc caggtccgat tgttcgttgc cgcgacctcc accggtgggg 17220 tcattgatcc tcggcggctg gcttgcggta gtgtcgtggt ggatgcagcg ttgccgaagg 17280 acatgcaacc tggctgggaa aaacgcgacg acattctggt cattgatggc ggcctggtct 17340 ctgcgactga cgcagtggac ttcggggcca tggcgctggg cctgggtcct aagcgcaata 17400 tcaatggttg cctggccgaa accatgatcc tggcgctgca gggtcgcgca gaggcattct 17460 ccatcgggcg cgaactgcct gctgagaagg ttctcgagat cggtcgcatc gccgagggac 17520 atggctttct gccttacccc atggcgagcg gcggcgaatc agtggatggc gctcggttcg 17580 atgaactgcg acgatttcac ggtgccaggc cgccagctgt cgtacacgac ctgaataccg 17640 gctcccagga gcttcgctca gaagtgctgc gctgcttcgg tacgcacatc aaccccattc 17700 tgcgtgaatt ctatgagttc aatcatgtcg agcgtgtctt cagcaatggc caaggctgct 17760 ggctgacgga tctggacggt cgccgttacc tggatttcgt agcgggatac ggctgcctca 17820 acacgggcca caaccatccg gagatagcag ccaggctgca ggaatacctc acccagcagc 17880 atcccacctt cgtgcagtac ctgtctgcgc cgttgcatgc cagcctgctg gccaaacgtc 17940 ttgccgagct ggctccagcc ggactggagc gcgtgttcct tagcaactct gggaccgaag 18000 ccgtggaggc ggctttgaag ctggctttgg ccgccagcga caaatccacg ctgctctact 18060 gcaccaatgg ctatcacggc aaaacgcttg gcgccctgtc cgtaacgggg cgtgagaagc 18120 accgcaaggc gttcgaaccg ttgctgccgc gctgcgagga gattccgttc gccgatgttt 18180 cggcgttgcg aaaccggttg ctcaaggggg atgtcscamc cttcatcatg gaaccgatcc 18240 agggtgaagg tggtgtcacc atggccccgg acggctatct cagagttgtg agggacctgt 18300 gctccgagca tgaatgcctc tggattctcg atgaaatcca gaccgggctc ggacgtaccg 18360 gcaagatgtt cgcctgcgaa tgggaagacg tctcgcccga catcatcgtg ctatccaaat 18420 ccctgtcggg tggtctggtg cctatcggng caacgctgtc ctcgaaagaa gtctggcaac 18480 gcgcctacgg caatatcgac cgattcgcat tgcacacctc gacgtttggc ggcgggaatt 18540 ttgctgccgc cgccgccatg gccgcgctgg acgtgatcga gcacgaagac ctgcccggca 18600 atgccgctct ggttggtgca cacctgcgac aagggctgga ggcgctggcc cgcaagcact 18660 atttcatcaa ggaggttcgc ggcgangcct gatatcgcca tcnaatttaa naacgatgtn 18720 tcaaatggca ttgaagcctt cgtgcgggat atgaccagcc ggatgcccgc caatgccgca 18780 gccacctacc gaatgatgcc tgccaaggcc cgcgaacacc tcgaagcagc catgcgcgaa 18840 ttggagtcca cgctggccga catgttcgtc ctgcgcatca tgaccaagtt gtcccaggag 18900 cacggcatcc tgaccttcgt gacggccaac aacaaccgtg tcatgcggat tcagccgccg 18960 ctggttctat cactggcaga agccgatcgg ttcataaagg cattgggcga ggtctgcgag 19020 gatctctcga cctttgagtc atgagttcag tcatcgggat gcgctgttga ttcgatctcc 19080 ccttcccagc ttataagggt gacgcaatga acgttgaaac ccatcgtcct gcctcgctgg 19140 ctggactgtc cgcgcttctg ttgatagcga tgggcatgcc gatgatgatc ttctatgcta 19200 tcggcatcct gggcccgcac ctggtngccg acttggggat ttcccgtcag caactgggct 19260 ggctgaccgc cagcaccttc ggactcgccg ccctgctgtc accctgggca ggcgcactgg 19320 tccaacgcat gggcactcgt gcggggctga tatgcatgtt cctgctggtg gggttgtcct 19380 tttcgctaat ggcggtcctg cctggcttcg gtggattggt cacggcatta ctgctttgcg 19440 ggacggccca gtcattggca aacccggcga ccaatcaggc catcgcgcat agcgtacccg 19500 ttgcgcggaa agcgggtgtc gtcggtctga agcagtcggg tgtgcaggcg tccgccttgt 19560 tggcgggcgt ggcgcttcca ccgctggtgc tgatgtgggg atggcgtggc gcgttggcag 19620 cctgggtgcc cgtggcattg gtcatggccg cattggtgac ctattgggta cctgcaaaat 19680 cggtgtcagc gccaagcctg ccattgcgcg tgcgtggacc caatgtgtgg ctgtcgatat 19740 tgatggccat tcagctttgc gcaggtcttg cgctgtcctc gttcatgacc ttcctcggcg 19800 tctatgccgc ccagatcggc gtatcggtca gcaccatcgg agccatggtc agttgctttg 19860 gcgccatggg gatcctgtcc cgcgtgctgc tgacaccgat cgctgacaag ctgaaggacg 19920 aaaccatttt gctcggcgtg ctgttcatac ttgcaggcct ggcgctggcg gtcatgcgcg 19980 aggccaacac tcagcagcac tggccactct ggcttggcgt gacgggaatg ggcctgaccg 20040 tggccgccag caatgccatc gccatgagca tgctgtngcg cgatgggcga tttggcggcg 20100 cagccacttc ggcggggatg ctatcggttg gattcttcgg cggcttcgcc gttggcccgc 20160 ctgcattcgg gtggttcttg gcccactcag agggcttcgc ggccgcctgg cttagcctga 20220 tcggtatctt ggtcgcaggc ggtcttctgt gcctgctgtt gctctacatg cgccatcggg 20280 aatgagtcca tgcaaggaaa taacctccat gaagttgcca tccgcgccat cttcaagcga 20340 atggatgaaa ttgccgcgca atgcggcgat cgtttcccgt tgttccgccg cagcgcagac 20400 ggactgtgga cgttatccac acgaggctct tggctgggtg ggttctgggc ggggctttgg 20460 tggcggcgcg ccgcctacac aggcagtgca gacgaccatg ctgtgncaga agcttggtcg 20520 gcgcgacttg ccccattgtt acgtnagcca tcgatcaatc gcagtttcgt tttctggtat 20580 ggcaccgcca tcggccatga gaagcgcggt aaagaccaag cgtcccagca acttgccaac 20640 caagcggctc tcgccatctc tgaaagtttc aaccccggat tgggtggctg gatgtccggt 20700 tccggcatgg gcgccggaga actcggaacg cgcgcgctca acgtcgacgc gctggcgccg 20760 acactggcac tgctgcacgg tcatggaggc cagaaagaac gcaaacgggc gcgtcagcat 20820 ttacagacct gccttcacta cctcgcaacc gagcatggcg cctggcgaac caatgctgtt 20880 ctagatgccg gtgacggcgc gaaacaggaa gcggccgggg cttggcctcg cggacaggcc 20940 tgggccatgc tgggcatggc tgaagcggtt cgattgtttg gagaagccta tcggcatccg 21000 gctttgcagg cttgcacgta ttggactgaa cgctggggag gcgccagtca gcctgcagaa 21060 acatcagggt tgcaacgccc gccgcaggac ctttccgcgc acgtgatttc ggcaactgcc 21120 atgctcaatc tgtcgcggct catcccaggc cagcattggt tgtaccgcca ggcacatatg 21180 caaatttctg cggtgcttga tgtcactgac gttgtcgaga cagggcaatt cgttgggcat 21240 ctttatcacg tcggcccaga tgatgaacag ctggttgagt cggcctgcgc gacattcttc 21300 ttgcttgaaa acctgattaa cctgtaacgc gctgtgctcg ttgcggttcg cgccgacgtc 21360 ccgaggggcc tgcccaacgc acaccgcatc tggcgctaca ggccgtcttc gcctctcacc 21420 gcggtcggcc ttcccccaaa cgcagcgccg tccctcgccc cagggcatct ggtgcgtatt 21480 agcgctggca ccatttcgcg ccactgaacg atacgctcag ggcctgagag gcttcaaagc 21540 agcgaaattc ctctttcgag caggcggcgc gcgatcatga tccgatgaat ttccgaggta 21600 ccatcgtaaa gccggaagac cctcgcatcg cggtagtagc gctcgatcgg caactcagtg 21660 caatagccca ttccgccaaa gatctgcact gcgctgtccg cgacgcggcc cagcatttcg 21720 gacgcgaaca acttcaccat cgaaaccttc tcacgcacat cccgcccttg atcgatttcc 21780 caggccgcgt tgagaagcat catcctggcg ccgtagattt cacactgcat gtccgccagc 21840 atcttctgca ccatctggaa actgccgatc ggggcaccga actgcgagcg ctgcttcgcg 21900 aaatcgaccg acatttccag tagtttcgaa gccatgccga cagccctggc accgacatag 21960 gccaaccgtg caacgttgag atggccaagt accagcgaca tgccccgccc cggctcgccg 22020 agcaattgtt gcggcgcgat ccggcagtcg gtgaagaaca gcccgtgctg atgggtaccg 22080 cgatgcccca tcatttcctg tatcggcccg agttcaaggc ccggcgtatc gcggtcgacc 22140 aaaaagcagg agataccttc ctcgctgcgt gcagtgacga tataagcgct cgcgatatcg 22200 gcatcgctga tgaagtgttt gctgccgttc aatatccact cgcaaccgtc gcgcttgacc 22260 gcggttctga tgccgtttgc gtccgaaccg gcctcgggct cgctcatggc aaccgacaca 22320 tgtatatcgc cacgcatggc ggcatgcagg tacttctcgc gctgctcgcc ggtacaatga 22380 acaaggatgc tgggaacgtg gccgaatgcc cgccttgcca acacatcctt ggttcggccg 22440 atctgctcat tgaccagaca gaattcaacg gcactcagcc cgccgccgcc cacctcctgc 22500 ggcatatgca tggcccacag ccccagctcc cgggcctttt gcttgaggcc ttcagccacg 22560 ctgggatcga tcgcattcgt tctttcgatc tcatcctcga gcggattgag ttcgttttcg 22620 acgaaccgct tcaccgtcga aaccagcatt tgcacttcat tgggaaggga aaaatcgatc 22680 atgtcgttgg cttccggcgt tccggccaag cgtgaaagcc ggcctgatgg atcaagttgc 22740 cagcgaggcg gtaactggcc caagggaaca ccacgtgctc ggcgacgctt gtgcgggtgg 22800 tgtagcagat gccgtaggtg acacgacaca aagacctgcc tacggtttcg tcgcgcacac 22860 gttgctcgct gcggatgatg gcaatgaacg cggagtcggc ctgcttttcc agatcggctc 22920 catcggctag tccgagggcc aaagacatca tcggcacacc agcacttgca ctggcgccaa 22980 tggaaacaag tcgctctcga aggtggcgac gttagcgaat cctgcggcga acaacgcctc 23040 ggggagttct ccctggtgcc agacatggtg gccacgcatc agcaacggca ggtagtacgc 23100 cagaaccgtg gctgtctgcg ccgccgtgag cggaatttca gcatgaatgc tcacgaaaac 23160 cccgcccggc gccagcgctg ctcggatctt acgtagcgtc tgcgcaagat ccggcacgaa 23220 gtgcaaaacc gaagcgcacc agatgaggtc ataaccgctg ccaatttcat cacgggtcag 23280 gtcgcctccc agcaccgaca gacgcgtgcc gagttgcgcc atctcgacgt tctgccgtgc 23340 cactgctgcg gttggcggca actcgaacgc agtgccatgg catccgggaa gagcgcgtgc 23400 caacgcaatg gccaccatgc cgggaccgca accgaggtcc aggaaacgtc gaatgccctt 23460 tagctccggc aggcgagcaa cgatcgcgag tgccgcttcg accgtaacag ccgattgctc 23520 ctgcacgatt tttgtccgag caagcgtggc ccagcattgg tcggtcggta ccggcgcctc 23580 ctcgatggca cgcacttgca gatattccgg caatcgcgtg ccaaagtcgc gtagcgaccg 23640 aaggcgaaag gcccaagcat caccgcagta atcggacgac tgcttggcga aaaaacgctt 23700 tgctttgtcg ctggaacgat agtgtggcgt cgacccagtg ctgtcatgga gctgaaaccg 23760 ctccaacaga tccatgctcc ataacaactc aagaacgggc tcgagcctgt tcggatcaag 23820 ttcgagctgc tctgcgattt tcccggcccc tgcctgttcg gtcagcacat cgaaaaggcc 23880 gctatccagc gcagtcgcca acgcatcagc ctgcacagac gccagcgcga gatcccagca 23940 ggattgcaat ggatgggcgt gttgaaattt catcttggac ctcataagcg ctacgtcacc 24000 cacaggccta cctcactcag caggctgttg atcgtggtcg cgccatcaat gccgatggtg 24060 tcgtggtttc gatacccagc gttatggaaa aaaaaaggac gactcgtgtg tccgacggtg 24120 caatccgcca tcagttgatc gcgtcgtatt cggcgccgcc gttgtcgaag gtctcgaact 24180 gcccatgtgt caatgcgccg aagctcgcca gcagcgcgtg aagtcaaaga cgtctggcat 24240 gaagctttat gcgaagtgtc accattgccc gctcccaaaa ggtgttttcg ccaaatcgct 24300 ctcgtcgcgc ggaggaatcc tgggcacttg aaccatgtgc tccatgggct tgacggccaa 24360 tccatggcag ataacgcagt caacaatagc aatatctcgc attcccattc gtattaggat 24420 cgctgtattt cgtatgcgat ccgtgcaact gggcgcgcgg cgcgagaaaa gctcgtacct 24480 ttaccgggct tcgatcattc gtcatcagca agcaacggca ggacgaacgc ggtggttgca 24540 gggcttttgc gggaagtgat aacggacgtt gtacaagcgt tcaggctcgc gtgcgcttga 24600 caccgctagg gctacaggga aatggacgat gccccgtaaa gtggcatttt cccttaccgg 24660 agatgggatc ggcggctctt cgctggccta accggaaact ccacagctcg cgcgctcacc 24720 ttaagcattg gccartgaaa cgcgcgcctc ggcgatttag cttcactgct artgcccggc 24780 atgcktaggc gcgtgaggtc gcggtggggt gttgtcgcgg agagtccgag gatgtgagtt 24840 agagcaacgg caggagcgcg tgcgcacagg ttaggcgtca cccggatagg ccgggtcgcg 24900 ctggtgtgct ccagatagat tcggtccagc acgtcttgac ctatgaagct gctttgggaa 24960 gccgcgacgt tggtagcttt gaccaatcgg ttccagaagc gatcgcagtg gaaaaagtgt 25020 tctgagggca gtgacctaag gctctatgtt agcaagagtc cttttacttt ctgcttgatt 25080 tcaactggac actctttaaa gccgtatttc ccggcattct ctttagatga gttaaatccc 25140 agatagtagc catcgtaaca aaaatagtaa ttgtcaatct ttcgcgtgct tggcttgtct 25200 ttttccagca tgtctagaag cgtctttaat tgtgagcgcg ttagccaaaa attttgaggt 25260 acagcagagt tgcattcata tgttcagctg taagagtaac ctagccataa agtttcttta 25320 acattcgcag tcaggtcacg cacgagaatc gttcgacttg ccgaaaccat agcgtgccag 25380 gaacttggta cggccgagct gcacgaattc atcgatggca gcctgcacgg cggcggggct 25440 acggagctgg gaaagctggg acaaaaacga ccacgtcctt atgttgtgta acgccgggaa 25500 ccctcaagct gcagctttct cgtttcgcag gccctggccc gatggttgcg ggccgggccg 25560 cacttacatc ggccttacgc tcaagtatga tcggcaaacc ctgccgccac caaggacaac 25620 ctcgtgccga tcgatgcctc gccgccctcc tcgctcgttg atgtagcgcc actggccgaa 25680 gcgctcgaac agttcgccga ggcgcgcaac tgggcgcagt tccactcgcc aaagaacctg 25740 gccatggccc ttgcgggcga gacgggcgaa ctcctcgaga tccgtcgacc tgcagccaag 25800 c 25801 <210> SEQ ID NO 14 <211> LENGTH: 1173 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1173) <223> OTHER INFORMATION: ORF F <400> SEQUENCE: 14 atg cca cta tca gcg ctg gtg gcg ccg gct ggg gaa ctg agc cgc gcc 48 Met Pro Leu Ser Ala Leu Val Ala Pro Ala Gly Glu Leu Ser Arg Ala 1 5 10 15 gag atc aac cgt tac agc cgc cac cta ctg ata ccc gat gtg ggc atg 96 Glu Ile Asn Arg Tyr Ser Arg His Leu Leu Ile Pro Asp Val Gly Met 20 25 30 atc ggg cag cgt cgg ttg aag aac gcc aag gtg ttg gtc atc ggc gcc 144 Ile Gly Gln Arg Arg Leu Lys Asn Ala Lys Val Leu Val Ile Gly Ala 35 40 45 ggc ggt ctt ggc tct ccg act ctg ctc tat cta gct gca gca ggt gtg 192 Gly Gly Leu Gly Ser Pro Thr Leu Leu Tyr Leu Ala Ala Ala Gly Val 50 55 60 ggc acc atc ggg ata atc gac ttt gat cgg gtt gac gac tcc aac ctt 240 Gly Thr Ile Gly Ile Ile Asp Phe Asp Arg Val Asp Asp Ser Asn Leu 65 70 75 80 cag cgc cag gtc atc cac ggg gtg gat acc gtg ggc gag ctc aag gtg 288 Gln Arg Gln Val Ile His Gly Val Asp Thr Val Gly Glu Leu Lys Val 85 90 95 gac agt gca aag aaa gcc att gcg cga cta aat ccc ttt gtc cag gtc 336 Asp Ser Ala Lys Lys Ala Ile Ala Arg Leu Asn Pro Phe Val Gln Val 100 105 110 gaa acc tat acc gat cgc ctg gaa cgg gac atg gcg atc gag ctg ttt 384 Glu Thr Tyr Thr Asp Arg Leu Glu Arg Asp Met Ala Ile Glu Leu Phe 115 120 125 tcg cgc tac gac ctg atc atg gac ggt acc gac aac ttc gca acc cgt 432 Ser Arg Tyr Asp Leu Ile Met Asp Gly Thr Asp Asn Phe Ala Thr Arg 130 135 140 tac ttg gtc aac gac gcc tgc gtg ctg gcc aac aaa ccc tat gtg tgg 480 Tyr Leu Val Asn Asp Ala Cys Val Leu Ala Asn Lys Pro Tyr Val Trp 145 150 155 160 ggc tcg ata ttc cgt ttc gaa ggg cag gcg tcc gtg ttc tgg gaa aac 528 Gly Ser Ile Phe Arg Phe Glu Gly Gln Ala Ser Val Phe Trp Glu Asn 165 170 175 gcc ccg aac gac ctg ggc ctg aac tac cgc gac ctg tat ccg gag cct 576 Ala Pro Asn Asp Leu Gly Leu Asn Tyr Arg Asp Leu Tyr Pro Glu Pro 180 185 190 ccg ccg ccc gag atg gcc ccc tcg tgc tcc gag ggc ggt gtg ttc ggc 624 Pro Pro Pro Glu Met Ala Pro Ser Cys Ser Glu Gly Gly Val Phe Gly 195 200 205 att ctt tgc gca tcc atc gca tcg atc atg gcc acc gag gcg gtc aag 672 Ile Leu Cys Ala Ser Ile Ala Ser Ile Met Ala Thr Glu Ala Val Lys 210 215 220 ctg atc acg ggc atc ggc gag cca cta ctg ggt cgg ctg gtg gtg tac 720 Leu Ile Thr Gly Ile Gly Glu Pro Leu Leu Gly Arg Leu Val Val Tyr 225 230 235 240 gac gct ttg gat atg cgc tat cgg gag ctt cct gtg cgc cgc ctg cca 768 Asp Ala Leu Asp Met Arg Tyr Arg Glu Leu Pro Val Arg Arg Leu Pro 245 250 255 aat cga caa ccg atc acc gac ctg gcc gag gac tat cag gtg ttc tgc 816 Asn Arg Gln Pro Ile Thr Asp Leu Ala Glu Asp Tyr Gln Val Phe Cys 260 265 270 ggt ctg ggg ttg ccc aaa ggt gac acg gcg gac gcc gtg cca ggg atc 864 Gly Leu Gly Leu Pro Lys Gly Asp Thr Ala Asp Ala Val Pro Gly Ile 275 280 285 agc gta acg gaa ctc aag aag cgg atg gat cag gac gag gtg cct gtg 912 Ser Val Thr Glu Leu Lys Lys Arg Met Asp Gln Asp Glu Val Pro Val 290 295 300 ctg ata gac gtg cgc gag ccc acc gag tgg gac atc gtc cgt att ccg 960 Leu Ile Asp Val Arg Glu Pro Thr Glu Trp Asp Ile Val Arg Ile Pro 305 310 315 320 ggc gca atc ttg gtg acc aaa tcg ccc acc gca gcg cag aca ctg cgc 1008 Gly Ala Ile Leu Val Thr Lys Ser Pro Thr Ala Ala Gln Thr Leu Arg 325 330 335 gag cga tac ggg gca gat gcc aac ctg gtg atc gtc tgc aag tcc ggg 1056 Glu Arg Tyr Gly Ala Asp Ala Asn Leu Val Ile Val Cys Lys Ser Gly 340 345 350 cgg cgc tct gcc gac gtg acc gcc gag ttg cta aac ctg ggc atg cgc 1104 Arg Arg Ser Ala Asp Val Thr Ala Glu Leu Leu Asn Leu Gly Met Arg 355 360 365 aat gtt cgc aac ctc gaa ggt ggc gtt ttg gcc tgg gtg aag gac gtg 1152 Asn Val Arg Asn Leu Glu Gly Gly Val Leu Ala Trp Val Lys Asp Val 370 375 380 gac tct tct ctg cct agc tac 1173 Asp Ser Ser Leu Pro Ser Tyr 385 390 <210> SEQ ID NO 15 <211> LENGTH: 391 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 15 Met Pro Leu Ser Ala Leu Val Ala Pro Ala Gly Glu Leu Ser Arg Ala 1 5 10 15 Glu Ile Asn Arg Tyr Ser Arg His Leu Leu Ile Pro Asp Val Gly Met 20 25 30 Ile Gly Gln Arg Arg Leu Lys Asn Ala Lys Val Leu Val Ile Gly Ala 35 40 45 Gly Gly Leu Gly Ser Pro Thr Leu Leu Tyr Leu Ala Ala Ala Gly Val 50 55 60 Gly Thr Ile Gly Ile Ile Asp Phe Asp Arg Val Asp Asp Ser Asn Leu 65 70 75 80 Gln Arg Gln Val Ile His Gly Val Asp Thr Val Gly Glu Leu Lys Val 85 90 95 Asp Ser Ala Lys Lys Ala Ile Ala Arg Leu Asn Pro Phe Val Gln Val 100 105 110 Glu Thr Tyr Thr Asp Arg Leu Glu Arg Asp Met Ala Ile Glu Leu Phe 115 120 125 Ser Arg Tyr Asp Leu Ile Met Asp Gly Thr Asp Asn Phe Ala Thr Arg 130 135 140 Tyr Leu Val Asn Asp Ala Cys Val Leu Ala Asn Lys Pro Tyr Val Trp 145 150 155 160 Gly Ser Ile Phe Arg Phe Glu Gly Gln Ala Ser Val Phe Trp Glu Asn 165 170 175 Ala Pro Asn Asp Leu Gly Leu Asn Tyr Arg Asp Leu Tyr Pro Glu Pro 180 185 190 Pro Pro Pro Glu Met Ala Pro Ser Cys Ser Glu Gly Gly Val Phe Gly 195 200 205 Ile Leu Cys Ala Ser Ile Ala Ser Ile Met Ala Thr Glu Ala Val Lys 210 215 220 Leu Ile Thr Gly Ile Gly Glu Pro Leu Leu Gly Arg Leu Val Val Tyr 225 230 235 240 Asp Ala Leu Asp Met Arg Tyr Arg Glu Leu Pro Val Arg Arg Leu Pro 245 250 255 Asn Arg Gln Pro Ile Thr Asp Leu Ala Glu Asp Tyr Gln Val Phe Cys 260 265 270 Gly Leu Gly Leu Pro Lys Gly Asp Thr Ala Asp Ala Val Pro Gly Ile 275 280 285 Ser Val Thr Glu Leu Lys Lys Arg Met Asp Gln Asp Glu Val Pro Val 290 295 300 Leu Ile Asp Val Arg Glu Pro Thr Glu Trp Asp Ile Val Arg Ile Pro 305 310 315 320 Gly Ala Ile Leu Val Thr Lys Ser Pro Thr Ala Ala Gln Thr Leu Arg 325 330 335 Glu Arg Tyr Gly Ala Asp Ala Asn Leu Val Ile Val Cys Lys Ser Gly 340 345 350 Arg Arg Ser Ala Asp Val Thr Ala Glu Leu Leu Asn Leu Gly Met Arg 355 360 365 Asn Val Arg Asn Leu Glu Gly Gly Val Leu Ala Trp Val Lys Asp Val 370 375 380 Asp Ser Ser Leu Pro Ser Tyr 385 390 <210> SEQ ID NO 16 <211> LENGTH: 1743 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1743) <223> OTHER INFORMATION: ORF J <400> SEQUENCE: 16 atg cct cac tcg caa gtc tta ctt ctc tgt caa cgc ccc ctg gag gtt 48 Met Pro His Ser Gln Val Leu Leu Leu Cys Gln Arg Pro Leu Glu Val 1 5 10 15 cct atg aac aac gct ggc atc atc gac ctg gtt cct gct gag gaa cgc 96 Pro Met Asn Asn Ala Gly Ile Ile Asp Leu Val Pro Ala Glu Glu Arg 20 25 30 caa cgt tgg gtg cag gac ggt acc tac ccg aac cag ccc gta ttc acg 144 Gln Arg Trp Val Gln Asp Gly Thr Tyr Pro Asn Gln Pro Val Phe Thr 35 40 45 ctg ttt gcc gcc aaa gcc gaa gcg cat ccc gac aag aag gcc gtg ctg 192 Leu Phe Ala Ala Lys Ala Glu Ala His Pro Asp Lys Lys Ala Val Leu 50 55 60 tcg ccg caa ggt gac gtg acc tac ggc gag ctc ctc gat gca gcc ctg 240 Ser Pro Gln Gly Asp Val Thr Tyr Gly Glu Leu Leu Asp Ala Ala Leu 65 70 75 80 cgg atg gct cac agc ctg cgt gat tcg ggg atc gtg gcc ggc gac gtg 288 Arg Met Ala His Ser Leu Arg Asp Ser Gly Ile Val Ala Gly Asp Val 85 90 95 gtg gct tac cag ctc acc aac cac tgg ttg tgc tgc gca atc gac ctg 336 Val Ala Tyr Gln Leu Thr Asn His Trp Leu Cys Cys Ala Ile Asp Leu 100 105 110 gca gtg gca gcg ctc ggt gcc atc gtc gcc ccc ttc cct ccg gga cgc 384 Ala Val Ala Ala Leu Gly Ala Ile Val Ala Pro Phe Pro Pro Gly Arg 115 120 125 ggc aag ctg gat atc caa tcg ctg gtt cgc cgc tgc gac gcg cga gcg 432 Gly Lys Leu Asp Ile Gln Ser Leu Val Arg Arg Cys Asp Ala Arg Ala 130 135 140 gtg atc gtc ccg caa gcg tac gaa ggc atc gat ctg tgc gag gtt atc 480 Val Ile Val Pro Gln Ala Tyr Glu Gly Ile Asp Leu Cys Glu Val Ile 145 150 155 160 gag tca ctg cgc ccc acc ctg cta tcc atg cgc cgc ctg att gtt cag 528 Glu Ser Leu Arg Pro Thr Leu Leu Ser Met Arg Arg Leu Ile Val Gln 165 170 175 ggc aag cct cgc gaa gga tgg att acg ctc gat gag ctg atg agc acc 576 Gly Lys Pro Arg Glu Gly Trp Ile Thr Leu Asp Glu Leu Met Ser Thr 180 185 190 gag ccg ctg gat ctc gcc agc cta ccc agg gtg tgc ccg aac tcg ccg 624 Glu Pro Leu Asp Leu Ala Ser Leu Pro Arg Val Cys Pro Asn Ser Pro 195 200 205 gtg cgt ctg ctg gtg tct tca ggc acc gag tcg gag ccc aag ctg gtg 672 Val Arg Leu Leu Val Ser Ser Gly Thr Glu Ser Glu Pro Lys Leu Val 210 215 220 gcg tac tcg cac aat gcg ttg gtt ggt ggt cgc ggg cgc ttc ctg cag 720 Ala Tyr Ser His Asn Ala Leu Val Gly Gly Arg Gly Arg Phe Leu Gln 225 230 235 240 cgc atc gcg tcc gat ggc gaa gat ttt cgc ggc atg tac ctc gtt ccg 768 Arg Ile Ala Ser Asp Gly Glu Asp Phe Arg Gly Met Tyr Leu Val Pro 245 250 255 ctg ggt tcg tcc ttc ggc tcc act gcc acc ttc ggt gtg ttg tgc tgg 816 Leu Gly Ser Ser Phe Gly Ser Thr Ala Thr Phe Gly Val Leu Cys Trp 260 265 270 ctg ggt ggt tcg ctg gtc gta ttg ccc aag ttc gac gtg gat gaa gcc 864 Leu Gly Gly Ser Leu Val Val Leu Pro Lys Phe Asp Val Asp Glu Ala 275 280 285 atc aag gcg att gcg gca ttt cgg ccg ggc ttc att ctc ggc gta ccc 912 Ile Lys Ala Ile Ala Ala Phe Arg Pro Gly Phe Ile Leu Gly Val Pro 290 295 300 acc atg ctg caa cgc atc gcc gct caa ccg gcg ttg gag agc atc gac 960 Thr Met Leu Gln Arg Ile Ala Ala Gln Pro Ala Leu Glu Ser Ile Asp 305 310 315 320 aaa tcc agc ctg cgt ggt ttg atc gtc ggc ggc tcg gtc atc gac gag 1008 Lys Ser Ser Leu Arg Gly Leu Ile Val Gly Gly Ser Val Ile Asp Glu 325 330 335 gcc acc gtg cgc aaa tgc cgt gat gcg ttt ggc tgc ggc ttc atc agc 1056 Ala Thr Val Arg Lys Cys Arg Asp Ala Phe Gly Cys Gly Phe Ile Ser 340 345 350 ctc tac ggt tcc gcc gac ggc gtg aac tgc cat aac acc ctg gac gac 1104 Leu Tyr Gly Ser Ala Asp Gly Val Asn Cys His Asn Thr Leu Asp Asp 355 360 365 ccc atc gaa gtt gtg ctg acc agc gtc ggc aag ccc aat ccg gcg gtc 1152 Pro Ile Glu Val Val Leu Thr Ser Val Gly Lys Pro Asn Pro Ala Val 370 375 380 tgc gcg att cgt ctg gtg gac gac gaa ggc cgg gag gtc cgg caa ggc 1200 Cys Ala Ile Arg Leu Val Asp Asp Glu Gly Arg Glu Val Arg Gln Gly 385 390 395 400 gag gtt ggc gaa atc acc gcc cgc ggg cca ttg act cca atg cag tac 1248 Glu Val Gly Glu Ile Thr Ala Arg Gly Pro Leu Thr Pro Met Gln Tyr 405 410 415 gtc aac gcg ccg gag ctg gac gag cgt tac cgc gac ccg caa ggc tgg 1296 Val Asn Ala Pro Glu Leu Asp Glu Arg Tyr Arg Asp Pro Gln Gly Trp 420 425 430 gtg aag acc gga gat ctg ggc tac atc aac gac aag ggt tat ctg gtc 1344 Val Lys Thr Gly Asp Leu Gly Tyr Ile Asn Asp Lys Gly Tyr Leu Val 435 440 445 cta gcc ggt cgc aag aag gac gtc atc atc cgt ggg ggc gcc aat atc 1392 Leu Ala Gly Arg Lys Lys Asp Val Ile Ile Arg Gly Gly Ala Asn Ile 450 455 460 agc ccg acc cag att gaa ggc ctg gtc atg gcg cat ccc gat gtc gtg 1440 Ser Pro Thr Gln Ile Glu Gly Leu Val Met Ala His Pro Asp Val Val 465 470 475 480 acc gtt gcg tgc att cct gtt ccc gat gat gat ctc ggg cag cgg gtg 1488 Thr Val Ala Cys Ile Pro Val Pro Asp Asp Asp Leu Gly Gln Arg Val 485 490 495 tgc ctt tgc gtc acc ttg cgc gag ggt gca gcg aag ttt tcc ctg aaa 1536 Cys Leu Cys Val Thr Leu Arg Glu Gly Ala Ala Lys Phe Ser Leu Lys 500 505 510 gcg atc acc gac ttc ctg cgc gaa ctg gga ctg gag gtg aac aag ctc 1584 Ala Ile Thr Asp Phe Leu Arg Glu Leu Gly Leu Glu Val Asn Lys Leu 515 520 525 ccc gag tac cta cgc ttc tac cgc gct ctg cct ctg aca ccg gcg gga 1632 Pro Glu Tyr Leu Arg Phe Tyr Arg Ala Leu Pro Leu Thr Pro Ala Gly 530 535 540 aag atc gat aaa aaa gcg cta acc gag gaa gcc cgc gag ctg ggc acc 1680 Lys Ile Asp Lys Lys Ala Leu Thr Glu Glu Ala Arg Glu Leu Gly Thr 545 550 555 560 tcg ggc att tgt ccc gct ggg ccg ggg cag tcg act ccc gag cgc agc 1728 Ser Gly Ile Cys Pro Ala Gly Pro Gly Gln Ser Thr Pro Glu Arg Ser 565 570 575 tta cgg gag tac gca 1743 Leu Arg Glu Tyr Ala 580 <210> SEQ ID NO 17 <211> LENGTH: 581 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 17 Met Pro His Ser Gln Val Leu Leu Leu Cys Gln Arg Pro Leu Glu Val 1 5 10 15 Pro Met Asn Asn Ala Gly Ile Ile Asp Leu Val Pro Ala Glu Glu Arg 20 25 30 Gln Arg Trp Val Gln Asp Gly Thr Tyr Pro Asn Gln Pro Val Phe Thr 35 40 45 Leu Phe Ala Ala Lys Ala Glu Ala His Pro Asp Lys Lys Ala Val Leu 50 55 60 Ser Pro Gln Gly Asp Val Thr Tyr Gly Glu Leu Leu Asp Ala Ala Leu 65 70 75 80 Arg Met Ala His Ser Leu Arg Asp Ser Gly Ile Val Ala Gly Asp Val 85 90 95 Val Ala Tyr Gln Leu Thr Asn His Trp Leu Cys Cys Ala Ile Asp Leu 100 105 110 Ala Val Ala Ala Leu Gly Ala Ile Val Ala Pro Phe Pro Pro Gly Arg 115 120 125 Gly Lys Leu Asp Ile Gln Ser Leu Val Arg Arg Cys Asp Ala Arg Ala 130 135 140 Val Ile Val Pro Gln Ala Tyr Glu Gly Ile Asp Leu Cys Glu Val Ile 145 150 155 160 Glu Ser Leu Arg Pro Thr Leu Leu Ser Met Arg Arg Leu Ile Val Gln 165 170 175 Gly Lys Pro Arg Glu Gly Trp Ile Thr Leu Asp Glu Leu Met Ser Thr 180 185 190 Glu Pro Leu Asp Leu Ala Ser Leu Pro Arg Val Cys Pro Asn Ser Pro 195 200 205 Val Arg Leu Leu Val Ser Ser Gly Thr Glu Ser Glu Pro Lys Leu Val 210 215 220 Ala Tyr Ser His Asn Ala Leu Val Gly Gly Arg Gly Arg Phe Leu Gln 225 230 235 240 Arg Ile Ala Ser Asp Gly Glu Asp Phe Arg Gly Met Tyr Leu Val Pro 245 250 255 Leu Gly Ser Ser Phe Gly Ser Thr Ala Thr Phe Gly Val Leu Cys Trp 260 265 270 Leu Gly Gly Ser Leu Val Val Leu Pro Lys Phe Asp Val Asp Glu Ala 275 280 285 Ile Lys Ala Ile Ala Ala Phe Arg Pro Gly Phe Ile Leu Gly Val Pro 290 295 300 Thr Met Leu Gln Arg Ile Ala Ala Gln Pro Ala Leu Glu Ser Ile Asp 305 310 315 320 Lys Ser Ser Leu Arg Gly Leu Ile Val Gly Gly Ser Val Ile Asp Glu 325 330 335 Ala Thr Val Arg Lys Cys Arg Asp Ala Phe Gly Cys Gly Phe Ile Ser 340 345 350 Leu Tyr Gly Ser Ala Asp Gly Val Asn Cys His Asn Thr Leu Asp Asp 355 360 365 Pro Ile Glu Val Val Leu Thr Ser Val Gly Lys Pro Asn Pro Ala Val 370 375 380 Cys Ala Ile Arg Leu Val Asp Asp Glu Gly Arg Glu Val Arg Gln Gly 385 390 395 400 Glu Val Gly Glu Ile Thr Ala Arg Gly Pro Leu Thr Pro Met Gln Tyr 405 410 415 Val Asn Ala Pro Glu Leu Asp Glu Arg Tyr Arg Asp Pro Gln Gly Trp 420 425 430 Val Lys Thr Gly Asp Leu Gly Tyr Ile Asn Asp Lys Gly Tyr Leu Val 435 440 445 Leu Ala Gly Arg Lys Lys Asp Val Ile Ile Arg Gly Gly Ala Asn Ile 450 455 460 Ser Pro Thr Gln Ile Glu Gly Leu Val Met Ala His Pro Asp Val Val 465 470 475 480 Thr Val Ala Cys Ile Pro Val Pro Asp Asp Asp Leu Gly Gln Arg Val 485 490 495 Cys Leu Cys Val Thr Leu Arg Glu Gly Ala Ala Lys Phe Ser Leu Lys 500 505 510 Ala Ile Thr Asp Phe Leu Arg Glu Leu Gly Leu Glu Val Asn Lys Leu 515 520 525 Pro Glu Tyr Leu Arg Phe Tyr Arg Ala Leu Pro Leu Thr Pro Ala Gly 530 535 540 Lys Ile Asp Lys Lys Ala Leu Thr Glu Glu Ala Arg Glu Leu Gly Thr 545 550 555 560 Ser Gly Ile Cys Pro Ala Gly Pro Gly Gln Ser Thr Pro Glu Arg Ser 565 570 575 Leu Arg Glu Tyr Ala 580 <210> SEQ ID NO 18 <211> LENGTH: 1884 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1884) <223> OTHER INFORMATION: ORF I <400> SEQUENCE: 18 ctg gcg att caa atc cag ggg caa gtg caa cgc ttt tgt ctc tcg gtt 48 Leu Ala Ile Gln Ile Gln Gly Gln Val Gln Arg Phe Cys Leu Ser Val 1 5 10 15 ttg aat atg ccg aca ctc tta aat gaa ttt tcc ctg ctg cat tca tcc 96 Leu Asn Met Pro Thr Leu Leu Asn Glu Phe Ser Leu Leu His Ser Ser 20 25 30 act tcg ttt ccg ccg aat tgg aat gaa ctg caa ctt agc ctg acg gaa 144 Thr Ser Phe Pro Pro Asn Trp Asn Glu Leu Gln Leu Ser Leu Thr Glu 35 40 45 cag gcc aga tta ttg ggc att tgc ccg ctc gca atc tcg ccg cct gtg 192 Gln Ala Arg Leu Leu Gly Ile Cys Pro Leu Ala Ile Ser Pro Pro Val 50 55 60 gat atg gaa gga gcc gca ttc cag ctg cag cat ccg gct att tct cct 240 Asp Met Glu Gly Ala Ala Phe Gln Leu Gln His Pro Ala Ile Ser Pro 65 70 75 80 att cag gcc cac ttc gcc tca cca gcc ggc tgg ctg cca aat cga cac 288 Ile Gln Ala His Phe Ala Ser Pro Ala Gly Trp Leu Pro Asn Arg His 85 90 95 ctc tcg gag ctg ctg ctg cag gcg ggc agc ggt ctt atg tcg gtg cac 336 Leu Ser Glu Leu Leu Leu Gln Ala Gly Ser Gly Leu Met Ser Val His 100 105 110 ggc cgt gct agc ggt agg gcc caa ccg ctg ggc gtg gat tat ctt tcg 384 Gly Arg Ala Ser Gly Arg Ala Gln Pro Leu Gly Val Asp Tyr Leu Ser 115 120 125 aca ctt acc gcc gtc atg acg ctg cac gga acg ctg gcc gca gcc gtg 432 Thr Leu Thr Ala Val Met Thr Leu His Gly Thr Leu Ala Ala Ala Val 130 135 140 ggg cag ctg cgt ggc ggt gca ttt gat cag gtt cag ctt tct cca ctg 480 Gly Gln Leu Arg Gly Gly Ala Phe Asp Gln Val Gln Leu Ser Pro Leu 145 150 155 160 gga tgc ggg ctg ctc agt atc ggg cag tat ctg gca ggc gcc acg gca 528 Gly Cys Gly Leu Leu Ser Ile Gly Gln Tyr Leu Ala Gly Ala Thr Ala 165 170 175 cca gaa gat cgt gag gcg ttc ctg ccg ggc ggc tcc gat ccg cat ttg 576 Pro Glu Asp Arg Glu Ala Phe Leu Pro Gly Gly Ser Asp Pro His Leu 180 185 190 agg ccg cca ttt cgt tcc gct gac ggc atc aca ttc gag ctg gaa acg 624 Arg Pro Pro Phe Arg Ser Ala Asp Gly Ile Thr Phe Glu Leu Glu Thr 195 200 205 ctc gac agc aca ccg tgk cga agc ttt tgg acc gcc gtc ggc att gaa 672 Leu Asp Ser Thr Pro Xaa Arg Ser Phe Trp Thr Ala Val Gly Ile Glu 210 215 220 tcg gaa ttg gcc ggt acg gcc tgg aaa ggt ttt ctg ctt cgc tac gcg 720 Ser Glu Leu Ala Gly Thr Ala Trp Lys Gly Phe Leu Leu Arg Tyr Ala 225 230 235 240 agg gcc gtg tcg cct cta cct gcc gcc tgt ctc acg gcg ctc gcc cgc 768 Arg Ala Val Ser Pro Leu Pro Ala Ala Cys Leu Thr Ala Leu Ala Arg 245 250 255 ctg cgt tac gca aag atc caa caa ttg gca gcg caa gcg ggt gtt gcg 816 Leu Arg Tyr Ala Lys Ile Gln Gln Leu Ala Ala Gln Ala Gly Val Ala 260 265 270 gtc gtg ccc gtc cgc acc gat gcg caa cgc cgc gag gac ccc gat tac 864 Val Val Pro Val Arg Thr Asp Ala Gln Arg Arg Glu Asp Pro Asp Tyr 275 280 285 cgg cag tca ctg gct acg cca tgg cag ttc gag tct ttc ccg ccg tcc 912 Arg Gln Ser Leu Ala Thr Pro Trp Gln Phe Glu Ser Phe Pro Pro Ser 290 295 300 ccc gaa agg cat cga gac acc gca ttt ccg tca ctg ctg ccg cta cag 960 Pro Glu Arg His Arg Asp Thr Ala Phe Pro Ser Leu Leu Pro Leu Gln 305 310 315 320 ggg atg cgc gtc atc gaa tcc tgt cga cgc att cag gga ccg ctg gcc 1008 Gly Met Arg Val Ile Glu Ser Cys Arg Arg Ile Gln Gly Pro Leu Ala 325 330 335 ggg cat ctg ctg gca tcg ctg ggc gcc gaa gtc att cgg ctg gag ccg 1056 Gly His Leu Leu Ala Ser Leu Gly Ala Glu Val Ile Arg Leu Glu Pro 340 345 350 ccg ggt ggc gat ccg ttg cga gcc atg ccg ccc tgc gcc gaa ggc tgt 1104 Pro Gly Gly Asp Pro Leu Arg Ala Met Pro Pro Cys Ala Glu Gly Cys 355 360 365 tcg gtg cgc ttt gac gcg ctg aac cac ctc aaa tcc gtt cac gaa gtc 1152 Ser Val Arg Phe Asp Ala Leu Asn His Leu Lys Ser Val His Glu Val 370 375 380 gat atc aaa tcc gcc cat ggg cgg cag ttg gtc tac gag ctc gcc cgc 1200 Asp Ile Lys Ser Ala His Gly Arg Gln Leu Val Tyr Glu Leu Ala Arg 385 390 395 400 gat gcg gat gtc ttt ctg cac aac tgg gcg ccc ggc aag gcc cat gaa 1248 Asp Ala Asp Val Phe Leu His Asn Trp Ala Pro Gly Lys Ala His Glu 405 410 415 atg caa ctg gat gct gaa cat ctg cgc agg gtt caa cca cat ctc gtt 1296 Met Gln Leu Asp Ala Glu His Leu Arg Arg Val Gln Pro His Leu Val 420 425 430 tac gcc tat gcg gga ggc tgg ggc cgg gct ccc gtc aat gcc ccg ggt 1344 Tyr Ala Tyr Ala Gly Gly Trp Gly Arg Ala Pro Val Asn Ala Pro Gly 435 440 445 acc gac ttc acc gtc cag gcc tgg tcg ggt gtg tcc gcc gcc att gca 1392 Thr Asp Phe Thr Val Gln Ala Trp Ser Gly Val Ser Ala Ala Ile Ala 450 455 460 cgt caa tcc ggc atc cgc ggc ggc tcg ctg ttc acc gtg ttg gat gtg 1440 Arg Gln Ser Gly Ile Arg Gly Gly Ser Leu Phe Thr Val Leu Asp Val 465 470 475 480 ctg ggc ggc gcg atc gcg gca ctg ggt gtg acg gcc gcg ttg ctc aat 1488 Leu Gly Gly Ala Ile Ala Ala Leu Gly Val Thr Ala Ala Leu Leu Asn 485 490 495 cga gca gtc acg ggc acg ggt act tat gtc gag agc tca ttg ctg ggc 1536 Arg Ala Val Thr Gly Thr Gly Thr Tyr Val Glu Ser Ser Leu Leu Gly 500 505 510 gcc gcc gat ctg ctg atg cac agc agc ggc aag gcg tcg agg ggc atc 1584 Ala Ala Asp Leu Leu Met His Ser Ser Gly Lys Ala Ser Arg Gly Ile 515 520 525 ttg tcc ggc gtg tat ccc acg cta tcg gga ctg atc gcc atc gac tgc 1632 Leu Ser Gly Val Tyr Pro Thr Leu Ser Gly Leu Ile Ala Ile Asp Cys 530 535 540 caa cac cca gat cag ttc cag tcg ctg gcc atg ttg ctg gac att cct 1680 Gln His Pro Asp Gln Phe Gln Ser Leu Ala Met Leu Leu Asp Ile Pro 545 550 555 560 gcc act gcg gat acc tgc cag cag acg ctg gcg gag cgc tta cgc aag 1728 Ala Thr Ala Asp Thr Cys Gln Gln Thr Leu Ala Glu Arg Leu Arg Lys 565 570 575 cga ccc gct tcg gaa tgg gaa acg gtg ctg aac gaa cgg ggc atc ggc 1776 Arg Pro Ala Ser Glu Trp Glu Thr Val Leu Asn Glu Arg Gly Ile Gly 580 585 590 gcc tgt gta gtc atc gaa gac ctc aag cag ctc gcc gcc gac acc cgc 1824 Ala Cys Val Val Ile Glu Asp Leu Lys Gln Leu Ala Ala Asp Thr Arg 595 600 605 atc tct gaa tgc ctc act cgc aag tct tac ttc tct gtc aac gcc ccc 1872 Ile Ser Glu Cys Leu Thr Arg Lys Ser Tyr Phe Ser Val Asn Ala Pro 610 615 620 tgg agg ttc cta 1884 Trp Arg Phe Leu 625 <210> SEQ ID NO 19 <211> LENGTH: 628 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 214 <223> OTHER INFORMATION: Xaa = unknown amino acid <400> SEQUENCE: 19 Leu Ala Ile Gln Ile Gln Gly Gln Val Gln Arg Phe Cys Leu Ser Val 1 5 10 15 Leu Asn Met Pro Thr Leu Leu Asn Glu Phe Ser Leu Leu His Ser Ser 20 25 30 Thr Ser Phe Pro Pro Asn Trp Asn Glu Leu Gln Leu Ser Leu Thr Glu 35 40 45 Gln Ala Arg Leu Leu Gly Ile Cys Pro Leu Ala Ile Ser Pro Pro Val 50 55 60 Asp Met Glu Gly Ala Ala Phe Gln Leu Gln His Pro Ala Ile Ser Pro 65 70 75 80 Ile Gln Ala His Phe Ala Ser Pro Ala Gly Trp Leu Pro Asn Arg His 85 90 95 Leu Ser Glu Leu Leu Leu Gln Ala Gly Ser Gly Leu Met Ser Val His 100 105 110 Gly Arg Ala Ser Gly Arg Ala Gln Pro Leu Gly Val Asp Tyr Leu Ser 115 120 125 Thr Leu Thr Ala Val Met Thr Leu His Gly Thr Leu Ala Ala Ala Val 130 135 140 Gly Gln Leu Arg Gly Gly Ala Phe Asp Gln Val Gln Leu Ser Pro Leu 145 150 155 160 Gly Cys Gly Leu Leu Ser Ile Gly Gln Tyr Leu Ala Gly Ala Thr Ala 165 170 175 Pro Glu Asp Arg Glu Ala Phe Leu Pro Gly Gly Ser Asp Pro His Leu 180 185 190 Arg Pro Pro Phe Arg Ser Ala Asp Gly Ile Thr Phe Glu Leu Glu Thr 195 200 205 Leu Asp Ser Thr Pro Xaa Arg Ser Phe Trp Thr Ala Val Gly Ile Glu 210 215 220 Ser Glu Leu Ala Gly Thr Ala Trp Lys Gly Phe Leu Leu Arg Tyr Ala 225 230 235 240 Arg Ala Val Ser Pro Leu Pro Ala Ala Cys Leu Thr Ala Leu Ala Arg 245 250 255 Leu Arg Tyr Ala Lys Ile Gln Gln Leu Ala Ala Gln Ala Gly Val Ala 260 265 270 Val Val Pro Val Arg Thr Asp Ala Gln Arg Arg Glu Asp Pro Asp Tyr 275 280 285 Arg Gln Ser Leu Ala Thr Pro Trp Gln Phe Glu Ser Phe Pro Pro Ser 290 295 300 Pro Glu Arg His Arg Asp Thr Ala Phe Pro Ser Leu Leu Pro Leu Gln 305 310 315 320 Gly Met Arg Val Ile Glu Ser Cys Arg Arg Ile Gln Gly Pro Leu Ala 325 330 335 Gly His Leu Leu Ala Ser Leu Gly Ala Glu Val Ile Arg Leu Glu Pro 340 345 350 Pro Gly Gly Asp Pro Leu Arg Ala Met Pro Pro Cys Ala Glu Gly Cys 355 360 365 Ser Val Arg Phe Asp Ala Leu Asn His Leu Lys Ser Val His Glu Val 370 375 380 Asp Ile Lys Ser Ala His Gly Arg Gln Leu Val Tyr Glu Leu Ala Arg 385 390 395 400 Asp Ala Asp Val Phe Leu His Asn Trp Ala Pro Gly Lys Ala His Glu 405 410 415 Met Gln Leu Asp Ala Glu His Leu Arg Arg Val Gln Pro His Leu Val 420 425 430 Tyr Ala Tyr Ala Gly Gly Trp Gly Arg Ala Pro Val Asn Ala Pro Gly 435 440 445 Thr Asp Phe Thr Val Gln Ala Trp Ser Gly Val Ser Ala Ala Ile Ala 450 455 460 Arg Gln Ser Gly Ile Arg Gly Gly Ser Leu Phe Thr Val Leu Asp Val 465 470 475 480 Leu Gly Gly Ala Ile Ala Ala Leu Gly Val Thr Ala Ala Leu Leu Asn 485 490 495 Arg Ala Val Thr Gly Thr Gly Thr Tyr Val Glu Ser Ser Leu Leu Gly 500 505 510 Ala Ala Asp Leu Leu Met His Ser Ser Gly Lys Ala Ser Arg Gly Ile 515 520 525 Leu Ser Gly Val Tyr Pro Thr Leu Ser Gly Leu Ile Ala Ile Asp Cys 530 535 540 Gln His Pro Asp Gln Phe Gln Ser Leu Ala Met Leu Leu Asp Ile Pro 545 550 555 560 Ala Thr Ala Asp Thr Cys Gln Gln Thr Leu Ala Glu Arg Leu Arg Lys 565 570 575 Arg Pro Ala Ser Glu Trp Glu Thr Val Leu Asn Glu Arg Gly Ile Gly 580 585 590 Ala Cys Val Val Ile Glu Asp Leu Lys Gln Leu Ala Ala Asp Thr Arg 595 600 605 Ile Ser Glu Cys Leu Thr Arg Lys Ser Tyr Phe Ser Val Asn Ala Pro 610 615 620 Trp Arg Phe Leu 625 <210> SEQ ID NO 20 <211> LENGTH: 357 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(357) <223> OTHER INFORMATION: ORF A <400> SEQUENCE: 20 gat ctg atc atc tgc gag gga cgc aat ctg cac ccg gag gac atc gag 48 Asp Leu Ile Ile Cys Glu Gly Arg Asn Leu His Pro Glu Asp Ile Glu 1 5 10 15 cac act gtg atc gag gcg ctg agc gac ttg cgg gcg caa agc tgc gca 96 His Thr Val Ile Glu Ala Leu Ser Asp Leu Arg Ala Gln Ser Cys Ala 20 25 30 gtg ttc agc cat gac gac gac cag cag cgc cag acc atc gtc gca gcc 144 Val Phe Ser His Asp Asp Asp Gln Gln Arg Gln Thr Ile Val Ala Ala 35 40 45 atc gaa ctg aat cgt gaa ctc aag cgc cgc ctg cag gac aat tgc cgc 192 Ile Glu Leu Asn Arg Glu Leu Lys Arg Arg Leu Gln Asp Asn Cys Arg 50 55 60 cag ctc aag gcc gtg gtg cgt agc gcg gtg gtg gac agt cat ggc atc 240 Gln Leu Lys Ala Val Val Arg Ser Ala Val Val Asp Ser His Gly Ile 65 70 75 80 acc ctc aac cgc atc gtc ttc gtg cag ccg acc agc att cac aag acg 288 Thr Leu Asn Arg Ile Val Phe Val Gln Pro Thr Ser Ile His Lys Thr 85 90 95 acc agc ggc aaa atc cag cgc gcg aag atg cgc cag ctc tat ctc gcc 336 Thr Ser Gly Lys Ile Gln Arg Ala Lys Met Arg Gln Leu Tyr Leu Ala 100 105 110 gag gaa ctg gac atg ctg caa 357 Glu Glu Leu Asp Met Leu Gln 115 <210> SEQ ID NO 21 <211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 21 Asp Leu Ile Ile Cys Glu Gly Arg Asn Leu His Pro Glu Asp Ile Glu 1 5 10 15 His Thr Val Ile Glu Ala Leu Ser Asp Leu Arg Ala Gln Ser Cys Ala 20 25 30 Val Phe Ser His Asp Asp Asp Gln Gln Arg Gln Thr Ile Val Ala Ala 35 40 45 Ile Glu Leu Asn Arg Glu Leu Lys Arg Arg Leu Gln Asp Asn Cys Arg 50 55 60 Gln Leu Lys Ala Val Val Arg Ser Ala Val Val Asp Ser His Gly Ile 65 70 75 80 Thr Leu Asn Arg Ile Val Phe Val Gln Pro Thr Ser Ile His Lys Thr 85 90 95 Thr Ser Gly Lys Ile Gln Arg Ala Lys Met Arg Gln Leu Tyr Leu Ala 100 105 110 Glu Glu Leu Asp Met Leu Gln 115 <210> SEQ ID NO 22 <211> LENGTH: 939 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(939) <223> OTHER INFORMATION: ORF B <400> SEQUENCE: 22 gtg aac tca agc gcc gcc tgc agg aca att gcc gcc agc tca agg ccg 48 Val Asn Ser Ser Ala Ala Cys Arg Thr Ile Ala Ala Ser Ser Arg Pro 1 5 10 15 tgg tgc gta gcg cgg tgg tgg aca gtc atg gca tca ccc tca acc gca 96 Trp Cys Val Ala Arg Trp Trp Thr Val Met Ala Ser Pro Ser Thr Ala 20 25 30 tcg tct tcg tgc agc cga cca gca ttc aca aga cga cca gcg gca aaa 144 Ser Ser Ser Cys Ser Arg Pro Ala Phe Thr Arg Arg Pro Ala Ala Lys 35 40 45 tcc agc gcg cga aga tgc gcc agc tct atc tcg ccg agg aac tgg aca 192 Ser Ser Ala Arg Arg Cys Ala Ser Ser Ile Ser Pro Arg Asn Trp Thr 50 55 60 tgc tgc aat gaa acc cgg tgc ccc ttc agc cct ggt gca gtg gct ggc 240 Cys Cys Asn Glu Thr Arg Cys Pro Phe Ser Pro Gly Ala Val Ala Gly 65 70 75 80 cga tgc ggc ggc acc agt gag cct ggt ctg ttt cca ttg cgc cgg cgg 288 Arg Cys Gly Gly Thr Ser Glu Pro Gly Leu Phe Pro Leu Arg Arg Arg 85 90 95 cag cgc gca aga gtt ttt tcc cgt gga aaa agg ccg ccc aag gcc ttt 336 Gln Arg Ala Arg Val Phe Ser Arg Gly Lys Arg Pro Pro Lys Ala Phe 100 105 110 gcg agc tgt atg ccg tcg aac tgc ccg gcc gca gct cgg cgc ttg cgc 384 Ala Ser Cys Met Pro Ser Asn Cys Pro Ala Ala Ala Arg Arg Leu Arg 115 120 125 gag ccg ttc gcc gag tcg ctg gcg caa ctg gcc gag gcc ttc gcc gag 432 Glu Pro Phe Ala Glu Ser Leu Ala Gln Leu Ala Glu Ala Phe Ala Glu 130 135 140 cag tgt cgc gcc ctg ccg aac aaa ccg ctt atc ctc ttc ggt cat agc 480 Gln Cys Arg Ala Leu Pro Asn Lys Pro Leu Ile Leu Phe Gly His Ser 145 150 155 160 ctg ggc gca ctg ctc gca tat gaa acc gct cgt gtg ttg ctg gcc aaa 528 Leu Gly Ala Leu Leu Ala Tyr Glu Thr Ala Arg Val Leu Leu Ala Lys 165 170 175 ggc gaa agg cca ccc gtg cag ctg ctg gtg tcc tcg cgc cag agc ccg 576 Gly Glu Arg Pro Pro Val Gln Leu Leu Val Ser Ser Arg Gln Ser Pro 180 185 190 gac tgg ctg ccg gcc tgc gcg ggt ctg ccg gcg cta aac gat cag gct 624 Asp Trp Leu Pro Ala Cys Ala Gly Leu Pro Ala Leu Asn Asp Gln Ala 195 200 205 ctg cgc gat tac cta ggc aac ctc gcc ggc acc ccg cca gag gtg ctg 672 Leu Arg Asp Tyr Leu Gly Asn Leu Ala Gly Thr Pro Pro Glu Val Leu 210 215 220 cag agc aag gcg atg atg gat ctc gcg gtg ccg gtg ctg cag gcc gac 720 Gln Ser Lys Ala Met Met Asp Leu Ala Val Pro Val Leu Gln Ala Asp 225 230 235 240 ctg aag ctg atc ctc aat tac cag cat cga cat ctc cag ccc ctg agt 768 Leu Lys Leu Ile Leu Asn Tyr Gln His Arg His Leu Gln Pro Leu Ser 245 250 255 atc ccg gtg ctg gtc ttc ggc gcg atc agc gac caa cag gtg cgc tat 816 Ile Pro Val Leu Val Phe Gly Ala Ile Ser Asp Gln Gln Val Arg Tyr 260 265 270 gag tcg ctg ctg agt tgg gag cgg atc agc ggc gag ggg ttc agc ctg 864 Glu Ser Leu Leu Ser Trp Glu Arg Ile Ser Gly Glu Gly Phe Ser Leu 275 280 285 cgg atg atc gag gga ggg cac ttt gcg gta atg cag caa ccg cag tgg 912 Arg Met Ile Glu Gly Gly His Phe Ala Val Met Gln Gln Pro Gln Trp 290 295 300 gtg ctt gac cag gtg caa acc gag tta 939 Val Leu Asp Gln Val Gln Thr Glu Leu 305 310 <210> SEQ ID NO 23 <211> LENGTH: 313 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 23 Val Asn Ser Ser Ala Ala Cys Arg Thr Ile Ala Ala Ser Ser Arg Pro 1 5 10 15 Trp Cys Val Ala Arg Trp Trp Thr Val Met Ala Ser Pro Ser Thr Ala 20 25 30 Ser Ser Ser Cys Ser Arg Pro Ala Phe Thr Arg Arg Pro Ala Ala Lys 35 40 45 Ser Ser Ala Arg Arg Cys Ala Ser Ser Ile Ser Pro Arg Asn Trp Thr 50 55 60 Cys Cys Asn Glu Thr Arg Cys Pro Phe Ser Pro Gly Ala Val Ala Gly 65 70 75 80 Arg Cys Gly Gly Thr Ser Glu Pro Gly Leu Phe Pro Leu Arg Arg Arg 85 90 95 Gln Arg Ala Arg Val Phe Ser Arg Gly Lys Arg Pro Pro Lys Ala Phe 100 105 110 Ala Ser Cys Met Pro Ser Asn Cys Pro Ala Ala Ala Arg Arg Leu Arg 115 120 125 Glu Pro Phe Ala Glu Ser Leu Ala Gln Leu Ala Glu Ala Phe Ala Glu 130 135 140 Gln Cys Arg Ala Leu Pro Asn Lys Pro Leu Ile Leu Phe Gly His Ser 145 150 155 160 Leu Gly Ala Leu Leu Ala Tyr Glu Thr Ala Arg Val Leu Leu Ala Lys 165 170 175 Gly Glu Arg Pro Pro Val Gln Leu Leu Val Ser Ser Arg Gln Ser Pro 180 185 190 Asp Trp Leu Pro Ala Cys Ala Gly Leu Pro Ala Leu Asn Asp Gln Ala 195 200 205 Leu Arg Asp Tyr Leu Gly Asn Leu Ala Gly Thr Pro Pro Glu Val Leu 210 215 220 Gln Ser Lys Ala Met Met Asp Leu Ala Val Pro Val Leu Gln Ala Asp 225 230 235 240 Leu Lys Leu Ile Leu Asn Tyr Gln His Arg His Leu Gln Pro Leu Ser 245 250 255 Ile Pro Val Leu Val Phe Gly Ala Ile Ser Asp Gln Gln Val Arg Tyr 260 265 270 Glu Ser Leu Leu Ser Trp Glu Arg Ile Ser Gly Glu Gly Phe Ser Leu 275 280 285 Arg Met Ile Glu Gly Gly His Phe Ala Val Met Gln Gln Pro Gln Trp 290 295 300 Val Leu Asp Gln Val Gln Thr Glu Leu 305 310 <210> SEQ ID NO 24 <211> LENGTH: 564 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(564) <223> OTHER INFORMATION: ORF D <400> SEQUENCE: 24 atg tct tac agg cca ggc act gca cgc ccg aga gga act gga cga cta 48 Met Ser Tyr Arg Pro Gly Thr Ala Arg Pro Arg Gly Thr Gly Arg Leu 1 5 10 15 ccc acg aag cca aac cct gtc gaa aca ctg ccg ttc cct tct ctc ctt 96 Pro Thr Lys Pro Asn Pro Val Glu Thr Leu Pro Phe Pro Ser Leu Leu 20 25 30 gct cga ccg cac gcg ctg caa tcg tgt tgg ccg acg cag ctg acc gag 144 Ala Arg Pro His Ala Leu Gln Ser Cys Trp Pro Thr Gln Leu Thr Glu 35 40 45 ccg ctg cgc gat ggc agg ccg tgt ccg gtc tca ccc gct cct gct gga 192 Pro Leu Arg Asp Gly Arg Pro Cys Pro Val Ser Pro Ala Pro Ala Gly 50 55 60 cgg cgg cag gga tgg gcc gat acc gcc cgg gtg aag atg gct cag gtg 240 Arg Arg Gln Gly Trp Ala Asp Thr Ala Arg Val Lys Met Ala Gln Val 65 70 75 80 cag gcg gtc agg gcc gac gtg tcc tgt gag att ctt cct cga cag ccc 288 Gln Ala Val Arg Ala Asp Val Ser Cys Glu Ile Leu Pro Arg Gln Pro 85 90 95 gtg ttt tcg ccg ata gga cgg aca ctg aac atg gac ggc atg ccc ggc 336 Val Phe Ser Pro Ile Gly Arg Thr Leu Asn Met Asp Gly Met Pro Gly 100 105 110 ttg gtg gcg gtc gca ctg gcg aag ccg aag ctg gaa gtg tcc ggt gta 384 Leu Val Ala Val Ala Leu Ala Lys Pro Lys Leu Glu Val Ser Gly Val 115 120 125 ggg ttc gag tgc ccg gcc gcc gcc gtc gtg gcg cag cag ccc atc gaa 432 Gly Phe Glu Cys Pro Ala Ala Ala Val Val Ala Gln Gln Pro Ile Glu 130 135 140 agc gtc gga ctt ccc gac cgt ctg agt gcg cgc agc ggc gat ctg gta 480 Ser Val Gly Leu Pro Asp Arg Leu Ser Ala Arg Ser Gly Asp Leu Val 145 150 155 160 cag gag gtc gag caa cgg gat gac gtc tgt ttt ccg ttc gct tcc gtc 528 Gln Glu Val Glu Gln Arg Asp Asp Val Cys Phe Pro Phe Ala Ser Val 165 170 175 gct ggc ctg ctc gcc cgc aaa gag gaa aac ctc tca 564 Ala Gly Leu Leu Ala Arg Lys Glu Glu Asn Leu Ser 180 185 <210> SEQ ID NO 25 <211> LENGTH: 188 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 25 Met Ser Tyr Arg Pro Gly Thr Ala Arg Pro Arg Gly Thr Gly Arg Leu 1 5 10 15 Pro Thr Lys Pro Asn Pro Val Glu Thr Leu Pro Phe Pro Ser Leu Leu 20 25 30 Ala Arg Pro His Ala Leu Gln Ser Cys Trp Pro Thr Gln Leu Thr Glu 35 40 45 Pro Leu Arg Asp Gly Arg Pro Cys Pro Val Ser Pro Ala Pro Ala Gly 50 55 60 Arg Arg Gln Gly Trp Ala Asp Thr Ala Arg Val Lys Met Ala Gln Val 65 70 75 80 Gln Ala Val Arg Ala Asp Val Ser Cys Glu Ile Leu Pro Arg Gln Pro 85 90 95 Val Phe Ser Pro Ile Gly Arg Thr Leu Asn Met Asp Gly Met Pro Gly 100 105 110 Leu Val Ala Val Ala Leu Ala Lys Pro Lys Leu Glu Val Ser Gly Val 115 120 125 Gly Phe Glu Cys Pro Ala Ala Ala Val Val Ala Gln Gln Pro Ile Glu 130 135 140 Ser Val Gly Leu Pro Asp Arg Leu Ser Ala Arg Ser Gly Asp Leu Val 145 150 155 160 Gln Glu Val Glu Gln Arg Asp Asp Val Cys Phe Pro Phe Ala Ser Val 165 170 175 Ala Gly Leu Leu Ala Arg Lys Glu Glu Asn Leu Ser 180 185 <210> SEQ ID NO 26 <211> LENGTH: 1113 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1113) <223> OTHER INFORMATION: ORF E <400> SEQUENCE: 26 ttg ttc cag ctt gcc gcg ctg ccg gcg ata ttc aaa ttc ctg ttg gct 48 Leu Phe Gln Leu Ala Ala Leu Pro Ala Ile Phe Lys Phe Leu Leu Ala 1 5 10 15 gtg ccg gtg cag cgt gtg cgc ctc ggg cgc gga cat ttc gtg cac tgg 96 Val Pro Val Gln Arg Val Arg Leu Gly Arg Gly His Phe Val His Trp 20 25 30 ttg ctg ttg ctc tgt gcg cta cta ctg gcg ctg tac tgg cta atc gga 144 Leu Leu Leu Leu Cys Ala Leu Leu Leu Ala Leu Tyr Trp Leu Ile Gly 35 40 45 cgg cat aat ctg atc ggc gat cgc ata atg ctg ttc gcg ctg acc ttc 192 Arg His Asn Leu Ile Gly Asp Arg Ile Met Leu Phe Ala Leu Thr Phe 50 55 60 gcc atc agc att gcc gcc acg tgg gcc gac att ccg cta aat gcg cta 240 Ala Ile Ser Ile Ala Ala Thr Trp Ala Asp Ile Pro Leu Asn Ala Leu 65 70 75 80 gcg gtg cag tgg ttg ccg cgt agt gaa cag ttg cgc gcc ggc agc atc 288 Ala Val Gln Trp Leu Pro Arg Ser Glu Gln Leu Arg Ala Gly Ser Ile 85 90 95 cgt tcc gca gcg ctg ttc gta ggc gcc att gtt ggc ggc ggc gtc atg 336 Arg Ser Ala Ala Leu Phe Val Gly Ala Ile Val Gly Gly Gly Val Met 100 105 110 atc atg gtg cag gcg cgc gtg ggc tgg cag gcc ccc ttc tgg ctg cta 384 Ile Met Val Gln Ala Arg Val Gly Trp Gln Ala Pro Phe Trp Leu Leu 115 120 125 ggg gtc gga ctg ctg att ggc gcc ctg ccc ttc ctg ctg ttg cgt aga 432 Gly Val Gly Leu Leu Ile Gly Ala Leu Pro Phe Leu Leu Leu Arg Arg 130 135 140 cac gcc gca ctg ccc gag cag gcc gag ccg cgc gag act aca gat cct 480 His Ala Ala Leu Pro Glu Gln Ala Glu Pro Arg Glu Thr Thr Asp Pro 145 150 155 160 cca ccg ggc gtg atg gcg gac tgg gca agc ttc ttc cac cag cca ggg 528 Pro Pro Gly Val Met Ala Asp Trp Ala Ser Phe Phe His Gln Pro Gly 165 170 175 gcg cgg caa tgg aca ttg ctg ctg ctg acc agt ttc ccc ttc ctc ggc 576 Ala Arg Gln Trp Thr Leu Leu Leu Leu Thr Ser Phe Pro Phe Leu Gly 180 185 190 gcg acg tgg ctg tac ctc aaa cct tta ttg ttg gac atg ggc atg cag 624 Ala Thr Trp Leu Tyr Leu Lys Pro Leu Leu Leu Asp Met Gly Met Gln 195 200 205 cta gag cgc gtg gcc ttc atc gtt ggc atc gtc ggc ggc acc gca ggc 672 Leu Glu Arg Val Ala Phe Ile Val Gly Ile Val Gly Gly Thr Ala Gly 210 215 220 gca ctg ttc agc ctg ctc ggc gga cag cta gtg caa atg ttg ggc ata 720 Ala Leu Phe Ser Leu Leu Gly Gly Gln Leu Val Gln Met Leu Gly Ile 225 230 235 240 gca cgg gcc att gcc tgg tac ctg ctg gcg gcg ctg ggc gcg ctg gca 768 Ala Arg Ala Ile Ala Trp Tyr Leu Leu Ala Ala Leu Gly Ala Leu Ala 245 250 255 ctt ttg acg ttc agc gtc tgg gcc caa ctg ggg gcg gca tgg ctg att 816 Leu Leu Thr Phe Ser Val Trp Ala Gln Leu Gly Ala Ala Trp Leu Ile 260 265 270 gcc agc gcc ctc tgc gtg gca gcc agc atg ggc gcc atc tcg gcg ctg 864 Ala Ser Ala Leu Cys Val Ala Ala Ser Met Gly Ala Ile Ser Ala Leu 275 280 285 atg ttc ggg ttg acc atg ttc ttc acc cga aat cgg cgc aac gcg tcg 912 Met Phe Gly Leu Thr Met Phe Phe Thr Arg Asn Arg Arg Asn Ala Ser 290 295 300 gac tat gcc ctg caa acc acc atg ttc acc gtt gcg cga ctg gcg gtg 960 Asp Tyr Ala Leu Gln Thr Thr Met Phe Thr Val Ala Arg Leu Ala Val 305 310 315 320 ccg atc gcc gcc ggg gtg ttg ctc gac cgg gtg ggc tac acc ggc atg 1008 Pro Ile Ala Ala Gly Val Leu Leu Asp Arg Val Gly Tyr Thr Gly Met 325 330 335 ctc ctg gca atg acc ctg gcg ctg ctg ctt tcc ttc gcg ctc gcc tgt 1056 Leu Leu Ala Met Thr Leu Ala Leu Leu Leu Ser Phe Ala Leu Ala Cys 340 345 350 cgg gtg cgg gaa aag gtg gaa tct tcg gca cag tcg ata ctc gag cac 1104 Arg Val Arg Glu Lys Val Glu Ser Ser Ala Gln Ser Ile Leu Glu His 355 360 365 gag cgg gtt 1113 Glu Arg Val 370 <210> SEQ ID NO 27 <211> LENGTH: 371 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 27 Leu Phe Gln Leu Ala Ala Leu Pro Ala Ile Phe Lys Phe Leu Leu Ala 1 5 10 15 Val Pro Val Gln Arg Val Arg Leu Gly Arg Gly His Phe Val His Trp 20 25 30 Leu Leu Leu Leu Cys Ala Leu Leu Leu Ala Leu Tyr Trp Leu Ile Gly 35 40 45 Arg His Asn Leu Ile Gly Asp Arg Ile Met Leu Phe Ala Leu Thr Phe 50 55 60 Ala Ile Ser Ile Ala Ala Thr Trp Ala Asp Ile Pro Leu Asn Ala Leu 65 70 75 80 Ala Val Gln Trp Leu Pro Arg Ser Glu Gln Leu Arg Ala Gly Ser Ile 85 90 95 Arg Ser Ala Ala Leu Phe Val Gly Ala Ile Val Gly Gly Gly Val Met 100 105 110 Ile Met Val Gln Ala Arg Val Gly Trp Gln Ala Pro Phe Trp Leu Leu 115 120 125 Gly Val Gly Leu Leu Ile Gly Ala Leu Pro Phe Leu Leu Leu Arg Arg 130 135 140 His Ala Ala Leu Pro Glu Gln Ala Glu Pro Arg Glu Thr Thr Asp Pro 145 150 155 160 Pro Pro Gly Val Met Ala Asp Trp Ala Ser Phe Phe His Gln Pro Gly 165 170 175 Ala Arg Gln Trp Thr Leu Leu Leu Leu Thr Ser Phe Pro Phe Leu Gly 180 185 190 Ala Thr Trp Leu Tyr Leu Lys Pro Leu Leu Leu Asp Met Gly Met Gln 195 200 205 Leu Glu Arg Val Ala Phe Ile Val Gly Ile Val Gly Gly Thr Ala Gly 210 215 220 Ala Leu Phe Ser Leu Leu Gly Gly Gln Leu Val Gln Met Leu Gly Ile 225 230 235 240 Ala Arg Ala Ile Ala Trp Tyr Leu Leu Ala Ala Leu Gly Ala Leu Ala 245 250 255 Leu Leu Thr Phe Ser Val Trp Ala Gln Leu Gly Ala Ala Trp Leu Ile 260 265 270 Ala Ser Ala Leu Cys Val Ala Ala Ser Met Gly Ala Ile Ser Ala Leu 275 280 285 Met Phe Gly Leu Thr Met Phe Phe Thr Arg Asn Arg Arg Asn Ala Ser 290 295 300 Asp Tyr Ala Leu Gln Thr Thr Met Phe Thr Val Ala Arg Leu Ala Val 305 310 315 320 Pro Ile Ala Ala Gly Val Leu Leu Asp Arg Val Gly Tyr Thr Gly Met 325 330 335 Leu Leu Ala Met Thr Leu Ala Leu Leu Leu Ser Phe Ala Leu Ala Cys 340 345 350 Arg Val Arg Glu Lys Val Glu Ser Ser Ala Gln Ser Ile Leu Glu His 355 360 365 Glu Arg Val 370 <210> SEQ ID NO 28 <211> LENGTH: 2229 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2229) <223> OTHER INFORMATION: ORF L <400> SEQUENCE: 28 atg gca gca agc caa aga gga tgc ggg cat atg cag caa gag aga cgg 48 Met Ala Ala Ser Gln Arg Gly Cys Gly His Met Gln Gln Glu Arg Arg 1 5 10 15 aat atc gga ata ctc ttg gtc agc cag gat gaa aaa ctg gcg ttg gac 96 Asn Ile Gly Ile Leu Leu Val Ser Gln Asp Glu Lys Leu Ala Leu Asp 20 25 30 ctg gac atg gta gtg gaa agc gta aac ggt ttg ctc agt cgc aat acc 144 Leu Asp Met Val Val Glu Ser Val Asn Gly Leu Leu Ser Arg Asn Thr 35 40 45 gat acg cct ttc gat ttg cac ccg aac gac gaa tgc ttt ccc tac cgc 192 Asp Thr Pro Phe Asp Leu His Pro Asn Asp Glu Cys Phe Pro Tyr Arg 50 55 60 cag atc ttc gct cag gcc tgt cga tac ttg cgc tca caa ccc agg gac 240 Gln Ile Phe Ala Gln Ala Cys Arg Tyr Leu Arg Ser Gln Pro Arg Asp 65 70 75 80 aag ctg ccc gcc ctg ttc tct cgg tgc ttc cac tca atg gca acc gcc 288 Lys Leu Pro Ala Leu Phe Ser Arg Cys Phe His Ser Met Ala Thr Ala 85 90 95 cgc cag gcg ctg tcg gcg ggt gat tgg acg ttg tcc ccg ctt gaa tgc 336 Arg Gln Ala Leu Ser Ala Gly Asp Trp Thr Leu Ser Pro Leu Glu Cys 100 105 110 gtg ttg ctc gat acg cgc cat gag gaa ccg gtc gat gac gac ccc ttc 384 Val Leu Leu Asp Thr Arg His Glu Glu Pro Val Asp Asp Asp Pro Phe 115 120 125 ctt gcg ctt acg ctg caa caa ccg ccg acc cgg ccc tcg ccc tgc tct 432 Leu Ala Leu Thr Leu Gln Gln Pro Pro Thr Arg Pro Ser Pro Cys Ser 130 135 140 gca atg ctg ctg tgc gag gaa agg gtg ctg ggt aaa tgg atg tgc cgg 480 Ala Met Leu Leu Cys Glu Glu Arg Val Leu Gly Lys Trp Met Cys Arg 145 150 155 160 ctg ggg ggc aac cgc ctg gtg cgg gtg ccg cat tcg aat ccc tgg caa 528 Leu Gly Gly Asn Arg Leu Val Arg Val Pro His Ser Asn Pro Trp Gln 165 170 175 cgc cgc gcc gat atc ctc cgg ctg att ctg gat cat ctg gag cat gcc 576 Arg Arg Ala Asp Ile Leu Arg Leu Ile Leu Asp His Leu Glu His Ala 180 185 190 cat ttc aac cgc atg ctt gct cgc gcg cgc cag aaa gcc gat ggc cag 624 His Phe Asn Arg Met Leu Ala Arg Ala Arg Gln Lys Ala Asp Gly Gln 195 200 205 gtg acg ctg gca caa aag att cat cac ctc atg act gaa cgc tgg ggt 672 Val Thr Leu Ala Gln Lys Ile His His Leu Met Thr Glu Arg Trp Gly 210 215 220 gat caa tgg gac ttc cac ttc tac acg ggc tcc atg gtc gcc ggt ttc 720 Asp Gln Trp Asp Phe His Phe Tyr Thr Gly Ser Met Val Ala Gly Phe 225 230 235 240 atc gac tcg atg aaa tct ctg ctg cag gga acg gac agc cac tgc ctg 768 Ile Asp Ser Met Lys Ser Leu Leu Gln Gly Thr Asp Ser His Cys Leu 245 250 255 acc ggt aac aac gag cac tca ttg gcc gta agc gcg ctg gct ggc tgg 816 Thr Gly Asn Asn Glu His Ser Leu Ala Val Ser Ala Leu Ala Gly Trp 260 265 270 cag ttg tat ggc cgt gcc tac gtc atc gcc atg acc tcc ggg atg atc 864 Gln Leu Tyr Gly Arg Ala Tyr Val Ile Ala Met Thr Ser Gly Met Ile 275 280 285 gac gaa gcg cgc ggg act ctg gcg aat ctc aag cgt gcc gca gcc cct 912 Asp Glu Ala Arg Gly Thr Leu Ala Asn Leu Lys Arg Ala Ala Ala Pro 290 295 300 ggc atc att gtc tgc gcc gac tcc ccg gaa acg atc tgg tat cca ttc 960 Gly Ile Ile Val Cys Ala Asp Ser Pro Glu Thr Ile Trp Tyr Pro Phe 305 310 315 320 cag ggt acg ctc gac gcc gac agc gac gga cat gcg gtc att gca gca 1008 Gln Gly Thr Leu Asp Ala Asp Ser Asp Gly His Ala Val Ile Ala Ala 325 330 335 cgc ggc ttg tgg cat ggg ttc atg cgt acc ccc gat gac atg cca gct 1056 Arg Gly Leu Trp His Gly Phe Met Arg Thr Pro Asp Asp Met Pro Ala 340 345 350 tgc ctt gca aat gcc ttc cag gcc ctt gat gag cgc ccc gct cca acc 1104 Cys Leu Ala Asn Ala Phe Gln Ala Leu Asp Glu Arg Pro Ala Pro Thr 355 360 365 ttc gtc ctt gca acg caa cac gtg ctg gag tcg caa gcg acc att tcg 1152 Phe Val Leu Ala Thr Gln His Val Leu Glu Ser Gln Ala Thr Ile Ser 370 375 380 gaa cca gtg gtg cag cgc ccc tcc tcc aag gct gca acg ctc tcc tgc 1200 Glu Pro Val Val Gln Arg Pro Ser Ser Lys Ala Ala Thr Leu Ser Cys 385 390 395 400 gcg caa cgc gaa cga ctg gat cag gcc gtg gcc gcc atc aac cac gac 1248 Ala Gln Arg Glu Arg Leu Asp Gln Ala Val Ala Ala Ile Asn His Asp 405 410 415 aac gcc cgg atg ctg tgg cat tgc ggt cgc ctc aca tcg gac gag cgg 1296 Asn Ala Arg Met Leu Trp His Cys Gly Arg Leu Thr Ser Asp Glu Arg 420 425 430 aat cag att ctt cgc ctg gcc gaa aaa gcc ggc atc gcc ctg gtg gac 1344 Asn Gln Ile Leu Arg Leu Ala Glu Lys Ala Gly Ile Ala Leu Val Asp 435 440 445 agc atc att cat ccg gga agc gtg ccc ggg ttc agc gac ggc aag tcc 1392 Ser Ile Ile His Pro Gly Ser Val Pro Gly Phe Ser Asp Gly Lys Ser 450 455 460 gta gcg aac tat ctg gga acg ctt tcc atg tat gga ttc aac cgc gcg 1440 Val Ala Asn Tyr Leu Gly Thr Leu Ser Met Tyr Gly Phe Asn Arg Ala 465 470 475 480 gtc tat gaa ttc ctg gaa gcg caa agc acc aac gaa gaa ggc gct ccc 1488 Val Tyr Glu Phe Leu Glu Ala Gln Ser Thr Asn Glu Glu Gly Ala Pro 485 490 495 tgg ctc ttt ttc ctc aag ggc aag gta gag caa tcg tct acc ccg tat 1536 Trp Leu Phe Phe Leu Lys Gly Lys Val Glu Gln Ser Ser Thr Pro Tyr 500 505 510 tcg gaa ggc aag ctc aag cgc aac ttc cgc att ggc caa gtc aac tgc 1584 Ser Glu Gly Lys Leu Lys Arg Asn Phe Arg Ile Gly Gln Val Asn Cys 515 520 525 aat gag gcg cac ctg tcg cca ttc acc ctg ctg gga ctg gac gtt cgg 1632 Asn Glu Ala His Leu Ser Pro Phe Thr Leu Leu Gly Leu Asp Val Arg 530 535 540 ttg gcg gac ttc ctg aat tac ctc gaa cca cgc ctg cag gtt gac gac 1680 Leu Ala Asp Phe Leu Asn Tyr Leu Glu Pro Arg Leu Gln Val Asp Asp 545 550 555 560 gcg gtg cta cgg cag cgc cgc gcc aga atc gaa caa ttg cgc aag ctg 1728 Ala Val Leu Arg Gln Arg Arg Ala Arg Ile Glu Gln Leu Arg Lys Leu 565 570 575 ccg gcc gct cag cca agc gac ctg atc gaa aca atg ccg atg acc ccg 1776 Pro Ala Ala Gln Pro Ser Asp Leu Ile Glu Thr Met Pro Met Thr Pro 580 585 590 aat tat ttc ttc cac cag ttg gga cga ctg gtg gtc gat ctg atc gag 1824 Asn Tyr Phe Phe His Gln Leu Gly Arg Leu Val Val Asp Leu Ile Glu 595 600 605 tcg cga agc tac cgc tat acc ggc gtc tac gac gtt ggt cgt tgc ggt 1872 Ser Arg Ser Tyr Arg Tyr Thr Gly Val Tyr Asp Val Gly Arg Cys Gly 610 615 620 ttg tcg gca atg cgc aac gtg gca cgc acc gat ccc ggt ttc tcc ggc 1920 Leu Ser Ala Met Arg Asn Val Ala Arg Thr Asp Pro Gly Phe Ser Gly 625 630 635 640 tgg tac ggt cgc gcc ctg atg ggt gac ggt ctg atg tcg ctg ccc tat 1968 Trp Tyr Gly Arg Ala Leu Met Gly Asp Gly Leu Met Ser Leu Pro Tyr 645 650 655 atc gcc ttg aag aac gaa cgc aat gtg ctg gca ttc atc ggt gat ggt 2016 Ile Ala Leu Lys Asn Glu Arg Asn Val Leu Ala Phe Ile Gly Asp Gly 660 665 670 gct cgc gca ata gtg ccg aat gtg gag cag cgc ctg gtt ggt tct ttc 2064 Ala Arg Ala Ile Val Pro Asn Val Glu Gln Arg Leu Val Gly Ser Phe 675 680 685 gat ccg ggg cat cgc agg gcg tgg tgg caa cgt cac cgt ttt cta tct 2112 Asp Pro Gly His Arg Arg Ala Trp Trp Gln Arg His Arg Phe Leu Ser 690 695 700 gag caa cgg cgt gct ttc cat gat tca gac cta tct gga caa acg cta 2160 Glu Gln Arg Arg Ala Phe His Asp Ser Asp Leu Ser Gly Gln Thr Leu 705 710 715 720 cac gct caa tgg ctg cag cca ggt caa cgt gcc att gac gga atg gaa 2208 His Ala Gln Trp Leu Gln Pro Gly Gln Arg Ala Ile Asp Gly Met Glu 725 730 735 gga ggc tcc cgt cga gca tgc 2229 Gly Gly Ser Arg Arg Ala Cys 740 <210> SEQ ID NO 29 <211> LENGTH: 743 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 29 Met Ala Ala Ser Gln Arg Gly Cys Gly His Met Gln Gln Glu Arg Arg 1 5 10 15 Asn Ile Gly Ile Leu Leu Val Ser Gln Asp Glu Lys Leu Ala Leu Asp 20 25 30 Leu Asp Met Val Val Glu Ser Val Asn Gly Leu Leu Ser Arg Asn Thr 35 40 45 Asp Thr Pro Phe Asp Leu His Pro Asn Asp Glu Cys Phe Pro Tyr Arg 50 55 60 Gln Ile Phe Ala Gln Ala Cys Arg Tyr Leu Arg Ser Gln Pro Arg Asp 65 70 75 80 Lys Leu Pro Ala Leu Phe Ser Arg Cys Phe His Ser Met Ala Thr Ala 85 90 95 Arg Gln Ala Leu Ser Ala Gly Asp Trp Thr Leu Ser Pro Leu Glu Cys 100 105 110 Val Leu Leu Asp Thr Arg His Glu Glu Pro Val Asp Asp Asp Pro Phe 115 120 125 Leu Ala Leu Thr Leu Gln Gln Pro Pro Thr Arg Pro Ser Pro Cys Ser 130 135 140 Ala Met Leu Leu Cys Glu Glu Arg Val Leu Gly Lys Trp Met Cys Arg 145 150 155 160 Leu Gly Gly Asn Arg Leu Val Arg Val Pro His Ser Asn Pro Trp Gln 165 170 175 Arg Arg Ala Asp Ile Leu Arg Leu Ile Leu Asp His Leu Glu His Ala 180 185 190 His Phe Asn Arg Met Leu Ala Arg Ala Arg Gln Lys Ala Asp Gly Gln 195 200 205 Val Thr Leu Ala Gln Lys Ile His His Leu Met Thr Glu Arg Trp Gly 210 215 220 Asp Gln Trp Asp Phe His Phe Tyr Thr Gly Ser Met Val Ala Gly Phe 225 230 235 240 Ile Asp Ser Met Lys Ser Leu Leu Gln Gly Thr Asp Ser His Cys Leu 245 250 255 Thr Gly Asn Asn Glu His Ser Leu Ala Val Ser Ala Leu Ala Gly Trp 260 265 270 Gln Leu Tyr Gly Arg Ala Tyr Val Ile Ala Met Thr Ser Gly Met Ile 275 280 285 Asp Glu Ala Arg Gly Thr Leu Ala Asn Leu Lys Arg Ala Ala Ala Pro 290 295 300 Gly Ile Ile Val Cys Ala Asp Ser Pro Glu Thr Ile Trp Tyr Pro Phe 305 310 315 320 Gln Gly Thr Leu Asp Ala Asp Ser Asp Gly His Ala Val Ile Ala Ala 325 330 335 Arg Gly Leu Trp His Gly Phe Met Arg Thr Pro Asp Asp Met Pro Ala 340 345 350 Cys Leu Ala Asn Ala Phe Gln Ala Leu Asp Glu Arg Pro Ala Pro Thr 355 360 365 Phe Val Leu Ala Thr Gln His Val Leu Glu Ser Gln Ala Thr Ile Ser 370 375 380 Glu Pro Val Val Gln Arg Pro Ser Ser Lys Ala Ala Thr Leu Ser Cys 385 390 395 400 Ala Gln Arg Glu Arg Leu Asp Gln Ala Val Ala Ala Ile Asn His Asp 405 410 415 Asn Ala Arg Met Leu Trp His Cys Gly Arg Leu Thr Ser Asp Glu Arg 420 425 430 Asn Gln Ile Leu Arg Leu Ala Glu Lys Ala Gly Ile Ala Leu Val Asp 435 440 445 Ser Ile Ile His Pro Gly Ser Val Pro Gly Phe Ser Asp Gly Lys Ser 450 455 460 Val Ala Asn Tyr Leu Gly Thr Leu Ser Met Tyr Gly Phe Asn Arg Ala 465 470 475 480 Val Tyr Glu Phe Leu Glu Ala Gln Ser Thr Asn Glu Glu Gly Ala Pro 485 490 495 Trp Leu Phe Phe Leu Lys Gly Lys Val Glu Gln Ser Ser Thr Pro Tyr 500 505 510 Ser Glu Gly Lys Leu Lys Arg Asn Phe Arg Ile Gly Gln Val Asn Cys 515 520 525 Asn Glu Ala His Leu Ser Pro Phe Thr Leu Leu Gly Leu Asp Val Arg 530 535 540 Leu Ala Asp Phe Leu Asn Tyr Leu Glu Pro Arg Leu Gln Val Asp Asp 545 550 555 560 Ala Val Leu Arg Gln Arg Arg Ala Arg Ile Glu Gln Leu Arg Lys Leu 565 570 575 Pro Ala Ala Gln Pro Ser Asp Leu Ile Glu Thr Met Pro Met Thr Pro 580 585 590 Asn Tyr Phe Phe His Gln Leu Gly Arg Leu Val Val Asp Leu Ile Glu 595 600 605 Ser Arg Ser Tyr Arg Tyr Thr Gly Val Tyr Asp Val Gly Arg Cys Gly 610 615 620 Leu Ser Ala Met Arg Asn Val Ala Arg Thr Asp Pro Gly Phe Ser Gly 625 630 635 640 Trp Tyr Gly Arg Ala Leu Met Gly Asp Gly Leu Met Ser Leu Pro Tyr 645 650 655 Ile Ala Leu Lys Asn Glu Arg Asn Val Leu Ala Phe Ile Gly Asp Gly 660 665 670 Ala Arg Ala Ile Val Pro Asn Val Glu Gln Arg Leu Val Gly Ser Phe 675 680 685 Asp Pro Gly His Arg Arg Ala Trp Trp Gln Arg His Arg Phe Leu Ser 690 695 700 Glu Gln Arg Arg Ala Phe His Asp Ser Asp Leu Ser Gly Gln Thr Leu 705 710 715 720 His Ala Gln Trp Leu Gln Pro Gly Gln Arg Ala Ile Asp Gly Met Glu 725 730 735 Gly Gly Ser Arg Arg Ala Cys 740 <210> SEQ ID NO 30 <211> LENGTH: 2292 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2292) <223> OTHER INFORMATION: ORF M <400> SEQUENCE: 30 atg ttg agt cag ccg agg cgc att gcg gag cgt gtc gtc gaa ggc gtc 48 Met Leu Ser Gln Pro Arg Arg Ile Ala Glu Arg Val Val Glu Gly Val 1 5 10 15 aat gcg tta aag gac gag ggg gca gaa ctc gta ggg ctg gga ggc ttc 96 Asn Ala Leu Lys Asp Glu Gly Ala Glu Leu Val Gly Leu Gly Gly Phe 20 25 30 acg tcc att gtt ggc aat cgc ggc ctg cag aca ctg gat cgc acg caa 144 Thr Ser Ile Val Gly Asn Arg Gly Leu Gln Thr Leu Asp Arg Thr Gln 35 40 45 att ccg gtg acg acg ggt aac tcg ctg act gcg tat gcc gcc tat atg 192 Ile Pro Val Thr Thr Gly Asn Ser Leu Thr Ala Tyr Ala Ala Tyr Met 50 55 60 aat ctg ttg ggt gtc cta tcc gcg ctg gaa att cca cca gaa aaa gcg 240 Asn Leu Leu Gly Val Leu Ser Ala Leu Glu Ile Pro Pro Glu Lys Ala 65 70 75 80 gag gtc gcc gtg ctc ggg tat ccc ggg tcg att gca ctg gcc atc gtt 288 Glu Val Ala Val Leu Gly Tyr Pro Gly Ser Ile Ala Leu Ala Ile Val 85 90 95 tgc ctg ctg gcg cca ttg ggg tgt cgg ctg cga ctg gtg cat cgc gga 336 Cys Leu Leu Ala Pro Leu Gly Cys Arg Leu Arg Leu Val His Arg Gly 100 105 110 ggg aaa cag cag gtc gac acg ttg ctt gag tac ctg ccc tca caa ttt 384 Gly Lys Gln Gln Val Asp Thr Leu Leu Glu Tyr Leu Pro Ser Gln Phe 115 120 125 cac gga caa gtg acg ctg cac gcc ggt ctg gaa gat tgc tat gcc cag 432 His Gly Gln Val Thr Leu His Ala Gly Leu Glu Asp Cys Tyr Ala Gln 130 135 140 gtc cga ttg ttt gtt gcc gcg acc tcc acc ggt ggg gtc att gat cct 480 Val Arg Leu Phe Val Ala Ala Thr Ser Thr Gly Gly Val Ile Asp Pro 145 150 155 160 cgg cgg ctg gct tgc ggt agt gtc gtg gtg gat gca gcg ttg ccg aag 528 Arg Arg Leu Ala Cys Gly Ser Val Val Val Asp Ala Ala Leu Pro Lys 165 170 175 gac atg caa cct ggc tgg gaa aaa cgc gac gac att ctg gtc att gat 576 Asp Met Gln Pro Gly Trp Glu Lys Arg Asp Asp Ile Leu Val Ile Asp 180 185 190 ggc ggc ctg gtc tct gcg act gac gca gtg gac ttc ggg gcc atg gcg 624 Gly Gly Leu Val Ser Ala Thr Asp Ala Val Asp Phe Gly Ala Met Ala 195 200 205 ctg ggc ctg ggt cct aag cgc aat atc aat ggt tgc ctg gcc gaa acc 672 Leu Gly Leu Gly Pro Lys Arg Asn Ile Asn Gly Cys Leu Ala Glu Thr 210 215 220 atg atc ctg gcg ctg cag ggt cgc gca gag gca ttc tcc atc ggg cgc 720 Met Ile Leu Ala Leu Gln Gly Arg Ala Glu Ala Phe Ser Ile Gly Arg 225 230 235 240 gaa ctg cct gct gag aag gtt ctc gag atc ggt cgc atc gcc gag gga 768 Glu Leu Pro Ala Glu Lys Val Leu Glu Ile Gly Arg Ile Ala Glu Gly 245 250 255 cat ggc ttt ctg cct tac ccc atg gcg agc ggc ggc gaa tca gtg gat 816 His Gly Phe Leu Pro Tyr Pro Met Ala Ser Gly Gly Glu Ser Val Asp 260 265 270 ggc gct cgg ttc gat gaa ctg cga cga ttt cac ggt gcc agg ccg cca 864 Gly Ala Arg Phe Asp Glu Leu Arg Arg Phe His Gly Ala Arg Pro Pro 275 280 285 gct gtc gta cac gac ctg aat acc ggc tcc cag gag ctt cgc tca gaa 912 Ala Val Val His Asp Leu Asn Thr Gly Ser Gln Glu Leu Arg Ser Glu 290 295 300 gtg ctg cgc tgc ttc ggt acg cac atc aac ccc att ctg cgt gaa ttc 960 Val Leu Arg Cys Phe Gly Thr His Ile Asn Pro Ile Leu Arg Glu Phe 305 310 315 320 tat gag ttc aat cat gtc gag cgt gtc ttc agc aat ggc caa ggc tgc 1008 Tyr Glu Phe Asn His Val Glu Arg Val Phe Ser Asn Gly Gln Gly Cys 325 330 335 tgg ctg acg gat ctg gac ggt cgc cgt tac ctg gat ttc gta gcg gga 1056 Trp Leu Thr Asp Leu Asp Gly Arg Arg Tyr Leu Asp Phe Val Ala Gly 340 345 350 tac ggc tgc ctc aac acg ggc cac aac cat ccg gag ata gca gcc agg 1104 Tyr Gly Cys Leu Asn Thr Gly His Asn His Pro Glu Ile Ala Ala Arg 355 360 365 ctg cag gaa tac ctc acc cag cag cat ccc acc ttc gtg cag tac ctg 1152 Leu Gln Glu Tyr Leu Thr Gln Gln His Pro Thr Phe Val Gln Tyr Leu 370 375 380 tct gcg ccg ttg cat gcc agc ctg ctg gcc aaa cgt ctt gcc gag ctg 1200 Ser Ala Pro Leu His Ala Ser Leu Leu Ala Lys Arg Leu Ala Glu Leu 385 390 395 400 gct cca gcc gga ctg gag cgc gtg ttc ctt agc aac tct ggg acc gaa 1248 Ala Pro Ala Gly Leu Glu Arg Val Phe Leu Ser Asn Ser Gly Thr Glu 405 410 415 gcc gtg gag gcg gct ttg aag ctg gct ttg gcc gcc agc gac aaa tcc 1296 Ala Val Glu Ala Ala Leu Lys Leu Ala Leu Ala Ala Ser Asp Lys Ser 420 425 430 acg ctg ctc tac tgc acc aat ggc tat cac ggc aaa acg ctt ggc gcc 1344 Thr Leu Leu Tyr Cys Thr Asn Gly Tyr His Gly Lys Thr Leu Gly Ala 435 440 445 ctg tcc gta acg ggg cgt gag aag cac cgc aag gcg ttc gaa ccg ttg 1392 Leu Ser Val Thr Gly Arg Glu Lys His Arg Lys Ala Phe Glu Pro Leu 450 455 460 ctg ccg cgc tgc gag gag att ccg ttc gcc gat gtt tcg gcg ttg cga 1440 Leu Pro Arg Cys Glu Glu Ile Pro Phe Ala Asp Val Ser Ala Leu Arg 465 470 475 480 aac cgg ttg ctc aag ggg gat gtc gca mcc ttc atc atg gaa ccg atc 1488 Asn Arg Leu Leu Lys Gly Asp Val Ala Xaa Phe Ile Met Glu Pro Ile 485 490 495 cag ggt gaa ggt ggt gtc acc atg gcc ccg gac ggc tat ctc aga gtt 1536 Gln Gly Glu Gly Gly Val Thr Met Ala Pro Asp Gly Tyr Leu Arg Val 500 505 510 gtg agg gac ctg tgc tcc gag cat gaa tgc ctc tgg att ctc gat gaa 1584 Val Arg Asp Leu Cys Ser Glu His Glu Cys Leu Trp Ile Leu Asp Glu 515 520 525 atc cag acc ggg ctc gga cgt acc ggc aag atg ttc gcc tgc gaa tgg 1632 Ile Gln Thr Gly Leu Gly Arg Thr Gly Lys Met Phe Ala Cys Glu Trp 530 535 540 gaa gac gtc tcg ccc gac atc atc gtg cta tcc aaa tcc ctg tcg ggt 1680 Glu Asp Val Ser Pro Asp Ile Ile Val Leu Ser Lys Ser Leu Ser Gly 545 550 555 560 ggt ctg gtg cct atc ggc gca acg ctg tcc tcg aaa gaa gtc tgg caa 1728 Gly Leu Val Pro Ile Gly Ala Thr Leu Ser Ser Lys Glu Val Trp Gln 565 570 575 cgc gcc tac ggc aat atc gac cga ttc gca ttg cac acc tcg acg ttt 1776 Arg Ala Tyr Gly Asn Ile Asp Arg Phe Ala Leu His Thr Ser Thr Phe 580 585 590 ggc ggc ggg aat ttt gct gcc gcc gcc gcc atg gcc gcg ctg gac gtg 1824 Gly Gly Gly Asn Phe Ala Ala Ala Ala Ala Met Ala Ala Leu Asp Val 595 600 605 atc gag cac gaa gac ctg ccc ggc aat gcc gct ctg gtt ggt gca cac 1872 Ile Glu His Glu Asp Leu Pro Gly Asn Ala Ala Leu Val Gly Ala His 610 615 620 ctg cga caa ggg ctg gag gcg ctg gcc cgc aag cac tat ttc atc aag 1920 Leu Arg Gln Gly Leu Glu Ala Leu Ala Arg Lys His Tyr Phe Ile Lys 625 630 635 640 gag gtt cgc ggg cga ggc ctg atg atc gcc atc gaa ttt cag aac gat 1968 Glu Val Arg Gly Arg Gly Leu Met Ile Ala Ile Glu Phe Gln Asn Asp 645 650 655 gtc tca aat ggc att gaa gcc ttc gtg cgg gat atg acc agc cgg atg 2016 Val Ser Asn Gly Ile Glu Ala Phe Val Arg Asp Met Thr Ser Arg Met 660 665 670 ccc gcc aat gcc gca gcc acc tac cga atg atg cct gcc aag gcc cgc 2064 Pro Ala Asn Ala Ala Ala Thr Tyr Arg Met Met Pro Ala Lys Ala Arg 675 680 685 gaa cac ctc gaa gca gcc atg cgc gaa ttg gag tcc acg ctg gcc gac 2112 Glu His Leu Glu Ala Ala Met Arg Glu Leu Glu Ser Thr Leu Ala Asp 690 695 700 atg ttc gtc ctg cgc atc atg acc aag ttg tcc cag gag cac ggc atc 2160 Met Phe Val Leu Arg Ile Met Thr Lys Leu Ser Gln Glu His Gly Ile 705 710 715 720 ctg acc ttc gtg acg gcc aac aac aac cgt gtc atg cgg att cag ccg 2208 Leu Thr Phe Val Thr Ala Asn Asn Asn Arg Val Met Arg Ile Gln Pro 725 730 735 ccg ctg gtt cta tca ctg gca gaa gcc gat cgg ttc ata aag gca ttg 2256 Pro Leu Val Leu Ser Leu Ala Glu Ala Asp Arg Phe Ile Lys Ala Leu 740 745 750 ggc gag gtc tgc gag gat ctc tcg acc ttt gag tca 2292 Gly Glu Val Cys Glu Asp Leu Ser Thr Phe Glu Ser 755 760 <210> SEQ ID NO 31 <211> LENGTH: 764 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 490 <223> OTHER INFORMATION: Xaa = unknown amino acid <400> SEQUENCE: 31 Met Leu Ser Gln Pro Arg Arg Ile Ala Glu Arg Val Val Glu Gly Val 1 5 10 15 Asn Ala Leu Lys Asp Glu Gly Ala Glu Leu Val Gly Leu Gly Gly Phe 20 25 30 Thr Ser Ile Val Gly Asn Arg Gly Leu Gln Thr Leu Asp Arg Thr Gln 35 40 45 Ile Pro Val Thr Thr Gly Asn Ser Leu Thr Ala Tyr Ala Ala Tyr Met 50 55 60 Asn Leu Leu Gly Val Leu Ser Ala Leu Glu Ile Pro Pro Glu Lys Ala 65 70 75 80 Glu Val Ala Val Leu Gly Tyr Pro Gly Ser Ile Ala Leu Ala Ile Val 85 90 95 Cys Leu Leu Ala Pro Leu Gly Cys Arg Leu Arg Leu Val His Arg Gly 100 105 110 Gly Lys Gln Gln Val Asp Thr Leu Leu Glu Tyr Leu Pro Ser Gln Phe 115 120 125 His Gly Gln Val Thr Leu His Ala Gly Leu Glu Asp Cys Tyr Ala Gln 130 135 140 Val Arg Leu Phe Val Ala Ala Thr Ser Thr Gly Gly Val Ile Asp Pro 145 150 155 160 Arg Arg Leu Ala Cys Gly Ser Val Val Val Asp Ala Ala Leu Pro Lys 165 170 175 Asp Met Gln Pro Gly Trp Glu Lys Arg Asp Asp Ile Leu Val Ile Asp 180 185 190 Gly Gly Leu Val Ser Ala Thr Asp Ala Val Asp Phe Gly Ala Met Ala 195 200 205 Leu Gly Leu Gly Pro Lys Arg Asn Ile Asn Gly Cys Leu Ala Glu Thr 210 215 220 Met Ile Leu Ala Leu Gln Gly Arg Ala Glu Ala Phe Ser Ile Gly Arg 225 230 235 240 Glu Leu Pro Ala Glu Lys Val Leu Glu Ile Gly Arg Ile Ala Glu Gly 245 250 255 His Gly Phe Leu Pro Tyr Pro Met Ala Ser Gly Gly Glu Ser Val Asp 260 265 270 Gly Ala Arg Phe Asp Glu Leu Arg Arg Phe His Gly Ala Arg Pro Pro 275 280 285 Ala Val Val His Asp Leu Asn Thr Gly Ser Gln Glu Leu Arg Ser Glu 290 295 300 Val Leu Arg Cys Phe Gly Thr His Ile Asn Pro Ile Leu Arg Glu Phe 305 310 315 320 Tyr Glu Phe Asn His Val Glu Arg Val Phe Ser Asn Gly Gln Gly Cys 325 330 335 Trp Leu Thr Asp Leu Asp Gly Arg Arg Tyr Leu Asp Phe Val Ala Gly 340 345 350 Tyr Gly Cys Leu Asn Thr Gly His Asn His Pro Glu Ile Ala Ala Arg 355 360 365 Leu Gln Glu Tyr Leu Thr Gln Gln His Pro Thr Phe Val Gln Tyr Leu 370 375 380 Ser Ala Pro Leu His Ala Ser Leu Leu Ala Lys Arg Leu Ala Glu Leu 385 390 395 400 Ala Pro Ala Gly Leu Glu Arg Val Phe Leu Ser Asn Ser Gly Thr Glu 405 410 415 Ala Val Glu Ala Ala Leu Lys Leu Ala Leu Ala Ala Ser Asp Lys Ser 420 425 430 Thr Leu Leu Tyr Cys Thr Asn Gly Tyr His Gly Lys Thr Leu Gly Ala 435 440 445 Leu Ser Val Thr Gly Arg Glu Lys His Arg Lys Ala Phe Glu Pro Leu 450 455 460 Leu Pro Arg Cys Glu Glu Ile Pro Phe Ala Asp Val Ser Ala Leu Arg 465 470 475 480 Asn Arg Leu Leu Lys Gly Asp Val Ala Xaa Phe Ile Met Glu Pro Ile 485 490 495 Gln Gly Glu Gly Gly Val Thr Met Ala Pro Asp Gly Tyr Leu Arg Val 500 505 510 Val Arg Asp Leu Cys Ser Glu His Glu Cys Leu Trp Ile Leu Asp Glu 515 520 525 Ile Gln Thr Gly Leu Gly Arg Thr Gly Lys Met Phe Ala Cys Glu Trp 530 535 540 Glu Asp Val Ser Pro Asp Ile Ile Val Leu Ser Lys Ser Leu Ser Gly 545 550 555 560 Gly Leu Val Pro Ile Gly Ala Thr Leu Ser Ser Lys Glu Val Trp Gln 565 570 575 Arg Ala Tyr Gly Asn Ile Asp Arg Phe Ala Leu His Thr Ser Thr Phe 580 585 590 Gly Gly Gly Asn Phe Ala Ala Ala Ala Ala Met Ala Ala Leu Asp Val 595 600 605 Ile Glu His Glu Asp Leu Pro Gly Asn Ala Ala Leu Val Gly Ala His 610 615 620 Leu Arg Gln Gly Leu Glu Ala Leu Ala Arg Lys His Tyr Phe Ile Lys 625 630 635 640 Glu Val Arg Gly Arg Gly Leu Met Ile Ala Ile Glu Phe Gln Asn Asp 645 650 655 Val Ser Asn Gly Ile Glu Ala Phe Val Arg Asp Met Thr Ser Arg Met 660 665 670 Pro Ala Asn Ala Ala Ala Thr Tyr Arg Met Met Pro Ala Lys Ala Arg 675 680 685 Glu His Leu Glu Ala Ala Met Arg Glu Leu Glu Ser Thr Leu Ala Asp 690 695 700 Met Phe Val Leu Arg Ile Met Thr Lys Leu Ser Gln Glu His Gly Ile 705 710 715 720 Leu Thr Phe Val Thr Ala Asn Asn Asn Arg Val Met Arg Ile Gln Pro 725 730 735 Pro Leu Val Leu Ser Leu Ala Glu Ala Asp Arg Phe Ile Lys Ala Leu 740 745 750 Gly Glu Val Cys Glu Asp Leu Ser Thr Phe Glu Ser 755 760 <210> SEQ ID NO 32 <211> LENGTH: 1539 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1539) <223> OTHER INFORMATION: ORF O <400> SEQUENCE: 32 gtg gcc acc atg tct ggc acc agg gag aac tcc ccg agg cgt tgt tcg 48 Val Ala Thr Met Ser Gly Thr Arg Glu Asn Ser Pro Arg Arg Cys Ser 1 5 10 15 ccg cag gat tcg cta acg tcg cca cct tcg aga gcg act tgt ttc cat 96 Pro Gln Asp Ser Leu Thr Ser Pro Pro Ser Arg Ala Thr Cys Phe His 20 25 30 tgg cgc cag tgc aag tgc tgg tgt gcc gat gat gtc ttt ggc cct cgg 144 Trp Arg Gln Cys Lys Cys Trp Cys Ala Asp Asp Val Phe Gly Pro Arg 35 40 45 act agc cga tgg agc cga tct gga aaa gca ggc cga ctc cgc gtt cat 192 Thr Ser Arg Trp Ser Arg Ser Gly Lys Ala Gly Arg Leu Arg Val His 50 55 60 tgc cat cat ccg cag cga gca acg tgt gcg cga cga aac cgt agg cag 240 Cys His His Pro Gln Arg Ala Thr Cys Ala Arg Arg Asn Arg Arg Gln 65 70 75 80 gtc ttt gtg tcg tgt cac cta cgg cat ctg cta cac cac ccg cac aag 288 Val Phe Val Ser Cys His Leu Arg His Leu Leu His His Pro His Lys 85 90 95 cgt cgc cga gca cgt ggt gtt ccc ttg ggc cag tta ccg cct cgc tgg 336 Arg Arg Arg Ala Arg Gly Val Pro Leu Gly Gln Leu Pro Pro Arg Trp 100 105 110 caa ctt gat cca tca ggc cgg ctt tca cgc ttg gcc gga acg ccg gaa 384 Gln Leu Asp Pro Ser Gly Arg Leu Ser Arg Leu Ala Gly Thr Pro Glu 115 120 125 gcc aac gac atg atc gat ttt tcc ctt ccc aat gaa gtg caa atg ctg 432 Ala Asn Asp Met Ile Asp Phe Ser Leu Pro Asn Glu Val Gln Met Leu 130 135 140 gtt tcg acg gtg aag cgg ttc gtc gaa aac gaa ctc aat ccg ctc gag 480 Val Ser Thr Val Lys Arg Phe Val Glu Asn Glu Leu Asn Pro Leu Glu 145 150 155 160 gat gag atc gaa aga acg aat gcg atc gat ccc agc gtg gct gaa ggc 528 Asp Glu Ile Glu Arg Thr Asn Ala Ile Asp Pro Ser Val Ala Glu Gly 165 170 175 ctc aag caa aag gcc cgg gag ctg ggg ctg tgg gcc atg cat atg ccg 576 Leu Lys Gln Lys Ala Arg Glu Leu Gly Leu Trp Ala Met His Met Pro 180 185 190 cag gag gtg ggc ggc ggc ggg ctg agt gcc gtt gaa ttc tgt ctg gtc 624 Gln Glu Val Gly Gly Gly Gly Leu Ser Ala Val Glu Phe Cys Leu Val 195 200 205 aat gag cag atc ggc cga acc aag gat gtg ttg gca agg cgg gca ttc 672 Asn Glu Gln Ile Gly Arg Thr Lys Asp Val Leu Ala Arg Arg Ala Phe 210 215 220 ggc cac gtt ccc agc atc ctt gtt cat tgt acc ggc gag cag cgc gag 720 Gly His Val Pro Ser Ile Leu Val His Cys Thr Gly Glu Gln Arg Glu 225 230 235 240 aag tac ctg cat gcc gcc atg cgt ggc gat ata cat gtg tcg gtt gcc 768 Lys Tyr Leu His Ala Ala Met Arg Gly Asp Ile His Val Ser Val Ala 245 250 255 atg agc gag ccc gag gcc ggt tcg gac gca aac ggc atc aga acc gcg 816 Met Ser Glu Pro Glu Ala Gly Ser Asp Ala Asn Gly Ile Arg Thr Ala 260 265 270 gtc aag cgc gac ggt tgc gag tgg ata ttg aac ggc agc aaa cac ttc 864 Val Lys Arg Asp Gly Cys Glu Trp Ile Leu Asn Gly Ser Lys His Phe 275 280 285 atc agc gat gcc gat atc gcg agc gct tat atc gtc act gca cgc agc 912 Ile Ser Asp Ala Asp Ile Ala Ser Ala Tyr Ile Val Thr Ala Arg Ser 290 295 300 gag gaa ggt atc tcc tgc ttt ttg gtc gac cgc gat acg ccg ggc ctt 960 Glu Glu Gly Ile Ser Cys Phe Leu Val Asp Arg Asp Thr Pro Gly Leu 305 310 315 320 gaa ctc ggg ccg ata cag gaa atg atg ggg cat cgc ggt acc cat cag 1008 Glu Leu Gly Pro Ile Gln Glu Met Met Gly His Arg Gly Thr His Gln 325 330 335 cac ggg ctg ttc ttc acc gac tgc cgg atc gcg ccg caa caa ttg ctc 1056 His Gly Leu Phe Phe Thr Asp Cys Arg Ile Ala Pro Gln Gln Leu Leu 340 345 350 ggc gag ccg ggg cgg ggc atg tcg ctg gta ctt ggc cat ctc aac gtt 1104 Gly Glu Pro Gly Arg Gly Met Ser Leu Val Leu Gly His Leu Asn Val 355 360 365 gca cgg ttg gcc tat gtc ggt gcc agg gct gtc ggc atg gct tcg aaa 1152 Ala Arg Leu Ala Tyr Val Gly Ala Arg Ala Val Gly Met Ala Ser Lys 370 375 380 cta ctg gaa atg tcg gtc gat ttc gcg aag cag cgc tcg cag ttc ggt 1200 Leu Leu Glu Met Ser Val Asp Phe Ala Lys Gln Arg Ser Gln Phe Gly 385 390 395 400 gcc ccg atc ggc agt ttc cag atg gtg cag aag atg ctg gcg gac atg 1248 Ala Pro Ile Gly Ser Phe Gln Met Val Gln Lys Met Leu Ala Asp Met 405 410 415 cag tgt gaa atc tac ggc gcc agg atg atg ctt ctc aac gcg gcc tgg 1296 Gln Cys Glu Ile Tyr Gly Ala Arg Met Met Leu Leu Asn Ala Ala Trp 420 425 430 gaa atc gat caa ggg cgg gat gtg cgt gag aag gtt tcg atg gtg aag 1344 Glu Ile Asp Gln Gly Arg Asp Val Arg Glu Lys Val Ser Met Val Lys 435 440 445 ttg ttc gcg tcc gaa atg ctg ggc cgc gtc gcg gac agc gca gtg cag 1392 Leu Phe Ala Ser Glu Met Leu Gly Arg Val Ala Asp Ser Ala Val Gln 450 455 460 atc ttt ggc gga atg ggc tat tgc act gag ttg ccg atc gag cgc tac 1440 Ile Phe Gly Gly Met Gly Tyr Cys Thr Glu Leu Pro Ile Glu Arg Tyr 465 470 475 480 tac cgc gat gcg agg gtc ttc cgg ctt tac gat ggt acc tcg gaa att 1488 Tyr Arg Asp Ala Arg Val Phe Arg Leu Tyr Asp Gly Thr Ser Glu Ile 485 490 495 cat cgg atc atg atc gcg cgc cgc ctg ctc gaa aga gga att tcg ctg 1536 His Arg Ile Met Ile Ala Arg Arg Leu Leu Glu Arg Gly Ile Ser Leu 500 505 510 ctt 1539 Leu <210> SEQ ID NO 33 <211> LENGTH: 513 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 33 Val Ala Thr Met Ser Gly Thr Arg Glu Asn Ser Pro Arg Arg Cys Ser 1 5 10 15 Pro Gln Asp Ser Leu Thr Ser Pro Pro Ser Arg Ala Thr Cys Phe His 20 25 30 Trp Arg Gln Cys Lys Cys Trp Cys Ala Asp Asp Val Phe Gly Pro Arg 35 40 45 Thr Ser Arg Trp Ser Arg Ser Gly Lys Ala Gly Arg Leu Arg Val His 50 55 60 Cys His His Pro Gln Arg Ala Thr Cys Ala Arg Arg Asn Arg Arg Gln 65 70 75 80 Val Phe Val Ser Cys His Leu Arg His Leu Leu His His Pro His Lys 85 90 95 Arg Arg Arg Ala Arg Gly Val Pro Leu Gly Gln Leu Pro Pro Arg Trp 100 105 110 Gln Leu Asp Pro Ser Gly Arg Leu Ser Arg Leu Ala Gly Thr Pro Glu 115 120 125 Ala Asn Asp Met Ile Asp Phe Ser Leu Pro Asn Glu Val Gln Met Leu 130 135 140 Val Ser Thr Val Lys Arg Phe Val Glu Asn Glu Leu Asn Pro Leu Glu 145 150 155 160 Asp Glu Ile Glu Arg Thr Asn Ala Ile Asp Pro Ser Val Ala Glu Gly 165 170 175 Leu Lys Gln Lys Ala Arg Glu Leu Gly Leu Trp Ala Met His Met Pro 180 185 190 Gln Glu Val Gly Gly Gly Gly Leu Ser Ala Val Glu Phe Cys Leu Val 195 200 205 Asn Glu Gln Ile Gly Arg Thr Lys Asp Val Leu Ala Arg Arg Ala Phe 210 215 220 Gly His Val Pro Ser Ile Leu Val His Cys Thr Gly Glu Gln Arg Glu 225 230 235 240 Lys Tyr Leu His Ala Ala Met Arg Gly Asp Ile His Val Ser Val Ala 245 250 255 Met Ser Glu Pro Glu Ala Gly Ser Asp Ala Asn Gly Ile Arg Thr Ala 260 265 270 Val Lys Arg Asp Gly Cys Glu Trp Ile Leu Asn Gly Ser Lys His Phe 275 280 285 Ile Ser Asp Ala Asp Ile Ala Ser Ala Tyr Ile Val Thr Ala Arg Ser 290 295 300 Glu Glu Gly Ile Ser Cys Phe Leu Val Asp Arg Asp Thr Pro Gly Leu 305 310 315 320 Glu Leu Gly Pro Ile Gln Glu Met Met Gly His Arg Gly Thr His Gln 325 330 335 His Gly Leu Phe Phe Thr Asp Cys Arg Ile Ala Pro Gln Gln Leu Leu 340 345 350 Gly Glu Pro Gly Arg Gly Met Ser Leu Val Leu Gly His Leu Asn Val 355 360 365 Ala Arg Leu Ala Tyr Val Gly Ala Arg Ala Val Gly Met Ala Ser Lys 370 375 380 Leu Leu Glu Met Ser Val Asp Phe Ala Lys Gln Arg Ser Gln Phe Gly 385 390 395 400 Ala Pro Ile Gly Ser Phe Gln Met Val Gln Lys Met Leu Ala Asp Met 405 410 415 Gln Cys Glu Ile Tyr Gly Ala Arg Met Met Leu Leu Asn Ala Ala Trp 420 425 430 Glu Ile Asp Gln Gly Arg Asp Val Arg Glu Lys Val Ser Met Val Lys 435 440 445 Leu Phe Ala Ser Glu Met Leu Gly Arg Val Ala Asp Ser Ala Val Gln 450 455 460 Ile Phe Gly Gly Met Gly Tyr Cys Thr Glu Leu Pro Ile Glu Arg Tyr 465 470 475 480 Tyr Arg Asp Ala Arg Val Phe Arg Leu Tyr Asp Gly Thr Ser Glu Ile 485 490 495 His Arg Ile Met Ile Ala Arg Arg Leu Leu Glu Arg Gly Ile Ser Leu 500 505 510 Leu <210> SEQ ID NO 34 <211> LENGTH: 159 <212> TYPE: DNA <213> ORGANISM: Pseudomonas stutzeri <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(159) <223> OTHER INFORMATION: ORF Q <400> SEQUENCE: 34 gtg ccg atc gat gcc tcg ccg ccc tcc tcg ctc gtt gat gta gcg cca 48 Val Pro Ile Asp Ala Ser Pro Pro Ser Ser Leu Val Asp Val Ala Pro 1 5 10 15 ctg gcc gaa gcg ctc gaa cag ttc gcc gag gcg cgc aac tgg gcg cag 96 Leu Ala Glu Ala Leu Glu Gln Phe Ala Glu Ala Arg Asn Trp Ala Gln 20 25 30 ttc cac tcg cca aag aac ctg gcc atg gcc ctt gcg ggc gag acg ggc 144 Phe His Ser Pro Lys Asn Leu Ala Met Ala Leu Ala Gly Glu Thr Gly 35 40 45 gaa ctc ctc gag atc 159 Glu Leu Leu Glu Ile 50 <210> SEQ ID NO 35 <211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400> SEQUENCE: 35 Val Pro Ile Asp Ala Ser Pro Pro Ser Ser Leu Val Asp Val Ala Pro 1 5 10 15 Leu Ala Glu Ala Leu Glu Gln Phe Ala Glu Ala Arg Asn Trp Ala Gln 20 25 30 Phe His Ser Pro Lys Asn Leu Ala Met Ala Leu Ala Gly Glu Thr Gly 35 40 45 Glu Leu Leu Glu Ile 50 

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
 1. An isolated nucleic acid molecule that is at least 70 percent identical to a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:13.
 2. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule is at least 90 percent identical to SEQ ID NO:1.
 3. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule is at least 90 percent identical to SEQ ID NO:3.
 4. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule is at least 90 percent identical to SEQ ID NO:5.
 5. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule is at least 90 percent identical to SEQ ID NO:7.
 6. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule is at least 90 percent identical to SEQ ID NO:13.
 7. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule consists of the nucleic acid sequence set forth in SEQ ID NO:1.
 8. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule consists of the nucleic acid sequence set forth in SEQ ID NO:3.
 9. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule consists of the nucleic acid sequence set forth in SEQ ID NO:5.
 10. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule consists of the nucleic acid sequence set forth in SEQ ID NO:7.
 11. An isolated nucleic acid molecule of claim 1 wherein said isolated nucleic acid molecule consists of the nucleic acid sequence set forth in SEQ ID NO:13.
 12. A vector comprising a nucleic acid molecule that is at least 70 percent identical to a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:13.
 13. A vector of claim 12 wherein said vector comprises a nucleic acid molecule that is at least 70 percent identical to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1.
 14. A vector of claim 12 wherein said vector comprises a nucleic acid molecule that is at least 70 percent identical to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:3.
 15. A vector of claim 12 wherein said vector comprises a nucleic acid molecule that is at least 70 percent identical to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:5.
 16. A vector of claim 12 wherein said vector comprises a nucleic acid molecule that is at least 70 percent identical to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:7.
 17. A vector of claim 12 wherein said vector comprises a nucleic acid molecule that is at least 70 percent identical to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:13.
 18. A host cell comprising a vector comprising a nucleic acid molecule that is at least 70 percent identical to a nucleic acid molecule consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:13.
 19. A host cell of claim 18 comprising a vector comprising a nucleic acid molecule that is at least 70 percent identical to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:1.
 20. A host cell of claim 18 comprising a vector comprising a nucleic acid molecule that is at least 70 percent identical to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:3.
 21. A host cell of claim 18 comprising a vector comprising a nucleic acid molecule that is at least 70 percent identical to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:5.
 22. A host cell of claim 18 comprising a vector comprising a nucleic acid molecule that is at least 70 percent identical to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:7.
 23. A host cell of claim 18 comprising a vector comprising a nucleic acid molecule that is at least 70 percent identical to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:13.
 24. A host cell of claim 23 wherein said host cell is a member of the genus Pseudomonas.
 25. A host cell of claim 23 wherein said host cell is a plant cell.
 26. An isolated nucleic acid molecule comprising: (a) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO: 1; (b) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO: 3; (c) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO: 5; (d) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO: 14; (e) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO: 16; and (f) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO:
 18. 27. An isolated nucleic acid molecule of claim 26 wherein said isolated nucleic acid molecule further comprises: (a) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO:7; (b) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO:9; and (c) a nucleic acid sequence that is at least 70 percent identical to the nucleic acid sequence set forth in SEQ ID NO:11.
 28. A vector comprising a nucleic acid molecule of claim
 26. 29. A host cell comprising a vector of claim
 28. 30. A host cell of claim 29 wherein said host cell is a member of the genus Pseudomonas.
 31. A host cell of claim 29 wherein said host cell is a plant cell.
 32. An isolated protein that is at least 70 percent identical to a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9.
 33. A method for reducing the amount of a metal in a substrate, said method comprising the steps of: (a) introducing into a substrate, said substrate comprising a metal ion species, a plant comprising roots and a PDTC gene cluster, the plant possessing a mechanism for transporting the metal ion species into the roots; and (b) expressing the PDTC gene cluster in the plant roots to form PDTC under conditions that enable the plant to remove an amount of the metal ion species from the substrate that is greater than the amount of the metal ion species that the plant would remove in the absence of expression of the PDTC gene cluster in the plant roots.
 34. A method for reducing the amount of a metal in the rhizosphere of a plant, said method comprising the steps of: (a) introducing into the rhizosphere of a plant at least one bacterial species that comprises a PDTC gene cluster, the rhizosphere comprising a metal ion species and the plant possessing a mechanism that transports the metal ion from the rhizosphere into the plant roots; and (b) culturing the at least one bacterial species in the rhizosphere for a time and under conditions that enable the bacterial species to synthesize PDTC and thereby increase availability of the metal ion to the plant roots so that the plant removes an amount of the metal ion from the substrate that is greater than the amount of the metal ion that the plant would remove if the bacterial species expressing PDTC was not present in the rhizosphere.
 35. A method for degrading carbon tetrachloride in a substrate, said method comprising the steps of: (a) introducing into a substrate a plant comprising roots and a PDTC gene cluster, the substrate further comprising Cu(II) ions and carbon tetrachloride that are introduced into the substrate, before, after or simultaneously with the introduction of the plant into the substrate; and (b) expressing the PDTC gene cluster in the plant roots under conditions that enable the plant to synthesize PDTC and release the PDTC into the substrate, thereby chemically degrading the carbon tetrachloride by the action of a complex formed between the PDTC and the Cu(II) ions.
 36. A method for degrading carbon tetrachloride in a substrate, said method comprising the steps of: (a) introducing into a substrate, comprising Cu(II) ions and carbon tetrachloride, at least one bacterial species that comprises a PDTC gene cluster under conditions that enable expression of the PDTC gene cluster to form PDTC; and (b) release of the PDTC into the substrate thereby chemically degrading the carbon tetrachloride by the action of a complex formed between the PDTC and the Cu(II) ions.
 37. A method for degrading carbon tetrachloride in a substrate, said method comprising the steps of: (a) introducing into a substrate, comprising carbon tetrachloride and Cu(II) ions, a nucleic acid molecule comprising a PDTC gene cluster comprising a plurality of PDTC biosynthetic genes, each of said PDTC biosynthetic genes being operably linked to at least one regulatory element that directs their expression within a microorganism, said nucleic acid molecule being attached to a particle; (b) uptake of the introduced nucleic acid molecule by a microorganism; (c) expression of the PDTC gene cluster within the microorganism to form PDTC; and (d) release of the PDTC into the substrate thereby chemically degrading the carbon tetrachloride by the action of a complex formed between the PDTC and the Cu(II) ions.
 38. A method for reducing the amount of a metal in a substrate, said method comprising the steps of: (a) contacting PDTC with a substrate comprising a metal ion species; and (b) allowing PDTC to form a metal complex with the metal ion species thereby reducing the amount of metal ion species in the substrate.
 39. The method of claim 38, wherein the substrate is water.
 40. The method of claim 38, wherein the substrate is soil.
 41. The method of claim 38, wherein the metal ion comprises a heavy metal ion.
 42. The method of claim 38, wherein the metal ion comprises a radionuclide.
 43. The method of claim 38, wherein the metal ion is selected from the group consisting of transition metals, lanthanide metals, actinide metals, radionuclides, and heavy metals.
 44. The method of claim 38 further comprising introducing a plant into the substrate, wherein the plant has an ability to take up the metal complex.
 45. A method for degrading carbon tetrachloride in a substrate, said method comprising the steps of: (a) contacting PDTC copper (II) complex with a substrate comprising carbon tetrachloride; and (b) chemically degrading the carbon tetrachloride by the action of the complex.
 46. The method of claim 45, wherein the substrate is water.
 47. The method of claim 45, wherein the substrate is soil.
 48. A method for immobilizing metal ions within a substrate, the method comprising the steps of: (a) contacting PDTC with a substrate to form a metal complex with the metal ion species; and (b) allowing PDTC to form a metal complex with the metal ion species thereby immobilizing the metal ion species 